U.S. patent number 5,654,183 [Application Number 08/188,286] was granted by the patent office on 1997-08-05 for genetically engineered mammalian neural crest stem cells.
This patent grant is currently assigned to California Institute of Technology. Invention is credited to David J. Anderson, Derek L. Stemple.
United States Patent |
5,654,183 |
Anderson , et al. |
August 5, 1997 |
Genetically engineered mammalian neural crest stem cells
Abstract
The invention includes mammalian multipotent neural stem cells
and their progeny and methods for the isolation and clonal
propagation of such cells. At the clonal level the stem cells are
capable of self regeneration and asymmetrical division. Lineage
restriction is demonstrated within developing clones which are
sensitive to the local environment. The invention also includes
such cells which are transfected with foreign nucleic acid, e.g.,
to produce an immortalized neural stem cell. The invention further
includes transplantation assays which allow for the identification
of mammalian multipotent neural stem cells from various tissues and
methods for transplanting mammalian neural stem cells and/or neural
or glial progenitors into mammals. A novel method for detecting
antibodies to neural cell surface markers is disclosed as well as a
monoclonal antibody to mouse LNGFR.
Inventors: |
Anderson; David J. (Altadena,
CA), Stemple; Derek L. (Newton, MA) |
Assignee: |
California Institute of
Technology (Pasadena, CA)
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Family
ID: |
27129806 |
Appl.
No.: |
08/188,286 |
Filed: |
January 28, 1994 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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996088 |
Dec 23, 1992 |
5365699 |
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920617 |
Jul 27, 1992 |
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Current U.S.
Class: |
435/456;
435/320.1; 435/325; 435/353; 435/368; 435/69.1 |
Current CPC
Class: |
C12N
5/0623 (20130101); C12N 2500/20 (20130101); C12N
2500/25 (20130101); C12N 2500/35 (20130101); C12N
2500/38 (20130101); C12N 2500/46 (20130101); C12N
2500/80 (20130101); C12N 2501/02 (20130101); C12N
2501/11 (20130101); C12N 2501/385 (20130101); C12N
2501/39 (20130101); C12N 2501/392 (20130101); C12N
2501/395 (20130101); C12N 2501/86 (20130101); C12N
2510/00 (20130101); C12N 2533/32 (20130101); C12N
2533/52 (20130101); C12N 2533/56 (20130101); C12N
2500/90 (20130101) |
Current International
Class: |
C12N
5/06 (20060101); C12N 015/85 (); C12N 015/00 () |
Field of
Search: |
;435/69.1,172.3,240.2,320.1 ;424/93.21 ;514/44 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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89/03872 |
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May 1989 |
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WO |
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93/01275 |
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Jan 1993 |
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WO |
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|
Primary Examiner: LeGuyader; John L.
Attorney, Agent or Firm: Flehr, Hohbach, Test, Albritton
& Herbert
Parent Case Text
This is a continuation-in-part of PCT application No.
PCT/US93/07000, filed Jul. 26, 1993, which is a
continuation-in-part of U.S. patent application Ser. No.
07/996,088, filed Dec. 23, 1992, now U.S. Pat. No. 5,365,699, which
is a continuation-in-part of U.S. patent application Ser. No.
07/920,617, filed Jul. 27, 1992, now abandoned.
Claims
What is claimed is:
1. An isolated cellular composition of genetically-engineered
mammalian neural crest stem cells which are capable of self-renewal
in a feeder cell-independent culture medium or glial cells, wherein
said genetically engineered neural crest stem cells express
low-affinity nerve growth factor receptor (LNGFR) and nestin, but
do not express neuronal or glial lineage markers including glial
fibrillary acidic protein (GFAP), wherein at least one neural crest
stem cell of said isolated composition is capable of
differentiation to a peripheral nervous system neuronal cell that
does not express LNGFR or nestin but does express
neurofilament-160, and wherein at least one neural crest stem cell
of said isolated composition is capable of differentiation to a
peripheral nervous system glial cell that expresses LNGFR, nestin
and GFAP.
2. The cellular composition of claim 1 wherein said neural crest
stem cells are isolated from the neural crest.
3. A cellular composition according to claims 1 wherein said neural
crest stem cells do not express sulfatide, myelin protein P.sub.o,
peripherin, high polysialic acid neural cell adhesion molecule
(high PSA-NCAM) or neurofilament.
4. A method for generating an isolated cellular composition of
genetically-engineered neural crest stem cells which express
low-affinity nerve growth factor receptor (LNGFR) and nestin but do
not express neuronal or glial lineage markers including glial
fibrillary acidic protein (GFAP), wherein at least one of said
neural crest stem cells is capable of differentiation to a
peripheral nervous system neuronal cell that does not express LNGFR
or nest in but does express neurofilament-160, and wherein at least
one of said neural crest stem cells is capable of differentiation
to a peripheral nervous system glial cell that expresses LNGFR,
nestin and GFAP, said method comprising contacting an isolated
neural crest stem cell with foreign nucleic acid under conditions
permissive for the uptake of said foreign nucleic acid into said
neural crest stem cell.
5. The method according to claim 4, wherein conditions permissive
for the uptake of said foreign nucleic acid comprise calcium
phosphate-mediated transfection.
6. The method according to claim 4, wherein conditions permissive
for the uptake of said foreign nucleic acid comprise retroviral
infection.
7. The method according to claim 4 wherein said neural crest stem
cells are isolated from the neural crest.
8. The method according to claim 4 wherein said neural crest stem
cells do not express sulfatide, myelin protein P.sub.o, peripherin,
high polysialic acid neural cell adhesion molecule (high PSA-NCAM)
or neurofilament.
9. A clonal population of genetically-engineered mammalian neural
crest stem cells which are capable of self-renewal in a feeder
cell-independent culture medium wherein said genetically engineered
neural crest stem cells express low-affinity nerve growth factor
receptor (LNGFR) and nestin, but do not express neuronal or glial
lineage markers including glial fibrillary acidic protein (GFAP),
wherein at least one of said stem cells of said clonal population
is capable of differentiation to a peripheral nervous system
neuronal cell that does not express LNGFR or nestin but does
express neurofilament-160, and wherein at least one of said stem
cells of said clonal population is capable of differentiation to a
peripheral nervous system glial cell that expresses LNGFR, nestin
and GFAP.
10. The clonal population of cells according to claim 9, wherein
said peripheral nervous system neuronal cells express
peripherin.
11. The clonal population of cells according to claim 9, wherein
said peripheral nervous system neuronal cells express
high-polysialic acid neural cell adhesion molecule (PSA-NCAM).
12. The clonal population of cells according to claim 9, wherein
said peripheral nervous system neuronal cells express
neurofilament-68.
13. The clonal population of cells according to claim 9, wherein
said peripheral nervous system neuronal cells express
neurofilament-160.
14. The clonal population of cells according to claim 9, wherein
said peripheral nervous system neuronal cells express
neurofilament-200.
15. The clonal population of cells according to claim 9, wherein
said peripheral nervous system glial cells express sulfatide.
16. The clonal population of cells according to claim 9, wherein
said peripheral nervous system glial cells express myelin protein
P.sub.o.
17. A clonal population of genetically-engineered mammalian neural
crest stem cells which are capable of self-renewal in a feeder
cell-independent culture medium wherein said genetically engineered
multipotent neural stem cells express low-affinity nerve growth
factor receptor (LNGFR) and nestin, but do not express neuronal
lineage or glial lineage markers including glial fibrillary acidic
protein (GFAP), sulfatide, myelin protein P.sub.o, peripherin,
high-polysialic acid neural cell adhesion molecule (PSA-NCAM), or
neurofilament-160, wherein at least one of said stem cells of said
clonal population is capable of differentiation to a peripheral
nervous system neuronal cell that does not express LNGFR or nestin
but does express peripherin, PSA-NCAM and neurofilament-160, and
wherein at least one of said stem cells of said clonal population
is capable of differentiation to a peripheral nervous system glial
cell finer expresses LNGFR, nestin, GFAP and sulfatide.
Description
FIELD OF THE INVENTION
The invention relates to the isolation, regeneration and use of
mammalian multipotent neural stem cells and progeny thereof.
BACKGROUND
The neural crest is a transient embryonic precursor population,
whose derivatives include cells having widely different
morphologies, characteristics and functions. These derivatives
include the neurons and glia of the entire peripheral nervous
system, melanocytes, cartilage and connective tissue of the head
and neck, stroma of various secretory glands and cells in the
outflow tract of the heart (for review, see Anderson, D. J. (1989)
Neuron 3:1-12). Much of the knowledge of the developmental
potential and fate of neural crest cells comes from studies in
avian systems. Fate maps have been established in aves and provide
evidence that several different crest cell derivatives may
originate from the same position along the neural tube (Le Dourain,
N. M. (1980) Nature 286:663-669). Schwann cells, melanocytes and
sensory and sympathetic neurons can all derive from the truncal
region of the neural tube. On the other hand, some derivatives were
found to originate from specific regions of the crest, e.g.,
enteric ganglia from the vagal and sacral regions. These studies
also revealed that the developmental potential of the neural crest
population at a given location along the neural tube is greater
than its developmental fate. This suggests that the new environment
encountered by the migrating crest cells influences their
developmental fate.
Single-cell lineage analysis in vivo, as well as clonal analysis in
vitro, have reportedly shown that early avian neural crest cells
are multipotential during, or shortly after, their detachment and
migration from the neural tube. In avian systems, certain clones
derived from single neural crest cells in culture were reported to
contain both catecholaminergic and pigmented cells (Sieber-Blum, M.
et al. (1980) Dev. Biol. 80:96-106). Baroffio, A. et al. (1988)
Proc. Natl. Acad. Sci. USA 85:5325-5329, reported that avian neural
crest cells from the cephalic region could generate clones which
gave rise to highly heterogeneous progeny when grown on
growth-arrested fibroblast feeder cell layers.
In vivo demonstration of the multipotency of early neural crest
cells was reported in chickens by Bronner-Fraser, M. et al. (1989)
Neuron 3:755-766. Individual neural crest cells, prior to their
migration from the neural tube, were injected with a fluorescent
dye. After 48 hours, the clonal progeny of injected cells were
found to reside in many or all of the locations to which neural
crest cells migrate, including sensory and sympathetic ganglia,
peripheral motor nerves and the skin. Phenotypic analysis of the
labelled cells revealed that at least some neural crest cells are
multipotent in vivo.
Following migration from the neural tube, these early multipotent
crest cells become segregated into different sublineages, which
generate restricted subsets of differentiated derivatives. The
mechanisms whereby neural crest cells become restricted to the
various sublineages are poorly understood. The fate of neural crest
derivatives is known to be controlled in some way by the embryonic
location in which their precursors come to reside (Le Douarin, N.
M. (1982) The Neural Crest., Cambridge University Press, Cambridge,
UK). The mechanism of specification for neural crest cells
derivatives is not known. In culture studies described above,
investigators reported that clones derived from primary neural
crest cells exhibited a mixture of phenotypes (Sieber-Blum, M. et
al. (1980) ibid; Baroffio, A. et al. (1988) ibid; Cohen, A. M. et
al. (1975) Dev. Biol. 46:262-280; Dupin, E. et al. (1990) Proc.
Natl. Acad. Sci. USA 87:1119-1123). Some clones contained only one
differentiated cell type whereas other clones contained many or all
of the assayable crest phenotypes.
The observation that apparently committed progenitors and
multipotent cells coexist in the neural crest may be interpreted to
reflect a pre-existing heterogeneity in the population of primary
crest cells or it may reflect asynchrony in a population of cells
that undergoes a progressive restriction in developmental
potential. Given the uncertainty in the art concerning the
developmental potential of neural crest cells, it is apparent that
a need exists for the isolation of neural crest cells in clonal
cultures. Although culture systems have been established which
allow the growth and differentiation of isolated avian neural crest
cells thereby permitting phenotypic identification of their
progeny, culture conditions which allow the self-renewal of
multipotent mammalian neural crest cells have not been reported.
Such culture conditions are essential for the isolation of
mammalian neural crest stem cells. Such stem cells are necessary in
order to understand how multipotent neural crest cells become
restricted to the various neural crest derivatives. In particular,
culture conditions which allow the growth and self-renewal of
mammalian neural crest stem cells are desirable so that the
particulars of the development of these mammalian stem cells may be
ascertained. This is desirable because a number of tumors of neural
crest derivatives exist in mammals, particularly humans. Knowledge
of mammalian neural crest stem cell development is therefore needed
to understand these disorders in humans. Additionally, the ability
to isolate and grow mammalian neural crest stem cells in vitro
allows for the possibility of using said stem cells to treat
peripheral neurological disorders in mammals, particularly
humans.
Accordingly, it is an object herein to provide clonal cultures of
mammalian multipotent neural stem cells and their progeny in feeder
cell-independent cultures. Another object of the invention is
directed to the demonstration that multipotential stem cells exist
in the neural crest. Another object of the invention is the
demonstration that these multipotent neural crest stem cells have
at least limited self regeneration capacity and undergo lineage
restriction in a manner that is sensitive to the local
environment.
A further object of the invention is to provide methods which allow
the growth and regeneration of multipotent neural stem cells in
feeder cell-independent cultures. Another object of the invention
is to provide methods which allow the differentiation of
multipotent neural crest stem cells into at least the progenitors
for, as well as, more differentiated neurons and glia of the
peripheral nervous system (PNS). A further object of the invention
is to provide methods which allow for the identification of
mammalian multipotent neural stem cells using transplantation
assays. Still further, an object of the invention is to provide
methods for transplanting neural crest stem cells or their progeny
into a mammal.
A further object of the invention is to extend the above methods to
provide clonal cultures of mammalian neural crest stem cells and
their progeny, to the detection or purification of glial or
neuronal progenitor cells, and to provide methods which allow the
growth, regeneration and differentiation of such cells from tissues
other than the embryonic neuronal crest. Still further, it is an
object herein to provide methods for transplanting progenitors of
such glial and neuronal cells and multipotent stem cell precursor
thereof into a mammal.
A further object of the invention is to provide cultures of
genetically-engineered multipotent neural stem cells and their
progeny. Still further, an object of the invention is to provide
methods for the generation of cultures of such
genetically-engineered multipotent neural stem cells and their
progeny including methods for immortalizing such cells.
Further, an object of the invention is to provide monoclonal
antibodies capable of recognizing surface markers which
characterize multipotent neural stem cells and/or their progeny. A
further object is to provide a novel procedure for screening sera
and hybridomas for such antibodies.
It is a further object of the invention to provide methods for
assaying the effects of various substances on neural stem cells.
Such effects include the differentiation of said cells into
neurons, glia or smooth muscle cells.
In addition, it as an object of the invention to provide methods
for producing mammalian smooth muscle cells including methods which
result in the preferential differential to smooth muscle cells at
the expense of other cell lineages.
SUMMARY OF THE INVENTION
In accordance with the forgoing objects, the invention includes the
isolation, clonal expansion and differentiation of mammalian
multipotent neural stem cells such as those derived from the neural
crest. The methods employ novel separation and culturing regimens
and bioassays for establishing the generation of multipotent neural
stem cells and their derivatives. These methods result in the
production of non-transformed neural stem cells and their progeny.
The invention demonstrates, at the clonal level, the self
regeneration and asymmetrical division of mammalian neural stem
cells for the first time in feeder cell-independent cultures.
Lineage restriction is demonstrated within a developing clone and
is shown to be sensitive to the local environment. For example,
neural crest stem cells cultured on a mixed substrate of
poly-D-lysine and fibronectin generate PNS neurons and glia, but on
fibronectin alone the stem cells generate PNS glia but not neurons.
The neurogenic potential of the neural crest stem cells, while not
expressed, is maintained over time on fibronectin. Therefore, both
the overt differentiation and maintenance of a latent developmental
potential of neural crest stem cells are shown to be sensitive to
the environment. The invention further includes transplantation
assays which allow for the identification of mammalian multipotent
neural stem cells from various tissues. It also includes methods
for transplanting mammalian neural stem cells and/or neural or
glial progenitors into mammals.
The invention also provides methods for obtaining a cellular
composition from mammalian tissue comprising one or more cells
having at least one property characteristic of a glial or neural
progenitor cell or a multipotent stem cell precursor of such cells.
The method comprises preparing a suspension comprising a population
of cells from a mammalian tissue; contacting the cell suspension
with a culture medium and substrate which permits self-renewal of
one or more of the glial or neural progenitor cells or multipotent
stem cell precursor, if present, in the cell suspension; and
identifying one or more such cells by its ability to self-renew and
differentiate feeder-cell independent culture.
The invention also includes alternate methods for obtaining a
cellular composition comprising one or more cells having at least
one property characteristic of a glial or neural progenitor cell or
a multipotent stem cell precursor thereof. The method comprises
preparing a suspension comprising cells from a mammalian tissue;
contacting the suspension with an antibody capable of forming a
complex with a neural cell-specific surface marker on said glial or
neural progenitor cells or multipotent stem cell precursor; and
isolating the complex, if formed, to obtain said cellular
composition.
The invention is also directed to cells made according to any of
the foregoing methods.
The invention also includes cultures of genetically-engineered
mammalian multipotent neural stem cells and their progeny. Nucleic
acid sequences encoding genes of interest are introduced into
multipotent neural stem cells where they are expressed. These genes
can include neurotrophic or survival factors and immortalizing
oncogenes. In addition, marker genes, such as the E. coli
.beta.-galactosidase gene, can be introduced to provide neural stem
cells and their progeny which can be identified based on the
expression of the marker gene. Selectable marker genes, such as the
neomycin phosphoribosyltransferase (neomycin-resistance, neo) or
hisD genes, may be introduced to provide for a population of
genetically-engineered stem cells which are identified by the
ability to grow in the presence of selective pressure (i.e., medium
containing neomycin or L-histidinol). Neural stem cells may be
transfected (genetically-engineered) with both a selectable marker
and a non-selectable marker to provide neural stem cells which
express both gene products.
The invention also includes methods for producing cultures of
genetically-engineered mammalian multipotent neural stem cells and
their progeny.
Still further, the invention includes methods for immortalizing
such cell lines by transfecting a glial or neural progenitor cell
or multipotent stem cell precursor thereof with a vector comprising
at least one immortalizing gene.
Further, the invention includes monoclonal antibodies capable of
recognizing surface markers characteristic of mammalian multipotent
neural stem cells and their progeny. The invention also includes a
method for screening hybridoma producing such monoclonal antibodies
which comprises contacting live neural cells with monoclonal
antibodies from a hybridoma and detecting whether the monoclonal
antibody binds to the neural cell.
In addition to the foregoing, the invention includes methods for
assaying the effects of substances on neural stem cells. Such
methods comprise contacting a culture of at least one neural stem
cell with a substance and determining the effect, if any, of the
substance on the differentiation of the neural stem cell. Such
differentiation can be to neurons, glial or smooth muscle cells or
a combination thereof.
The invention also includes methods for producing mammalian smooth
muscle cells comprising culturing at least one mammalian neural
stem cell under conditions which permit differentiation to smooth
muscle cells. Such conditions can result in a heterogeneous
population which includes smooth muscle cells and neurons and/or
glia. In alternate embodiments, factors instructive for smooth
muscle differentiation are used which result in the preferential
differentiation to smooth muscle at the expense of other cell
lineages.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A depicts the migration of rat neural crest cells from the
neural tube.
FIG. 1B demonstrates the expression of LNGFR and nestin by neural
crest cells.
FIGS. 1C and 1D show the FACS profile from neural crest cells
stained with anti-LNGFR (1D) and a control showing the background
staining of the secondary antibody (1C).
FIG. 2 demonstrates the clonal expansion of LNGFR.sup.+,
nestin.sup.+ rat neural crest cells.
FIG. 3 is a flow chart summarizing experiments demonstrating the
multipotency of mammalian neural crest cells.
FIG. 4 demonstrates the expression of neuronal traits in clones
derived from LNGFR.sup.+ founder cells.
FIG. 5 demonstrates the expression of Schwann cell phenotype by
neural crest-derived glia.
FIG. 6 shows the expression of peripherin, GFAP, and O.sub.4 in a
clone derived from a LNGFR.sup.+ founder cell.
FIG. 7 is a flow chart summarizing experiments demonstrating the
self-renewal of mammalian neural crest cells.
FIG. 8 demonstrates the self-renewal of multipotent neural crest
cells.
FIG. 9 demonstrates the multipotency of secondary founder
cells.
FIG. 10 provides a flow chart summarizing experiments demonstrating
the substrate effect on the fate of mammalian neural crest
cells.
FIG. 11 demonstrates that the neuronal differentiation of
multipotent neural crest cells is affected by their substrate.
FIG. 12 summarizes the percentage of different clone types which
result when founder cells are grown on either FN or FN/PDL
substrates.
FIG. 13 provides a flow chart summarizing experiments demonstrating
the instructive effect of the substrate on neural crest cell
fate.
FIG. 14 summarizes the percentage of the different clone types
which result when founder cells are treated with a PDL lysine
overlay at 48 hours (panel A) or day 5 (panel B).
FIG. 15 demonstrates the genetic-engineering of a multipotent
neural stem cell. Panel A depicts the expression of E. coli
.beta.-galactosidase (lacZ) in neural crest stem cells following
infection with a lacZ-containing retrovirus.
.beta.-galactosidase.sup.+ cells are indicated by the solid arrows.
Panel B depicts neural crest stem cells in phase contrast, in the
same microscopic field as shown in Panel A. Cells which do not
express .beta.-galactosidase are indicated by open arrows.
FIG. 16 demonstrates the specificity of a supernatant from a
hybridoma culture producing monoclonal antibody specific to mouse
LNGFR. Supernatants were screened using live Schwann cells isolated
from mouse sciatic nerve. Panel A shows that most cells are stained
with anti-LNGFR antibody (red staining; open arrows). Panel B shows
Schwann cell nuclei counter stained with DAPI. Comparison with
Panel A reveals a few cells not labeled by anti-LNGFR antibody
(blue staining; open arrows).
FIGS. 17 A and B depict the identification of smooth muscle cells
in neural crest cultures. Cultures of neural crest stem cells were
fixed and double-labeled with antibodies to p75-LNGFR (FIG. 17B,
orange staining), and SMA (FIG. 17B, green staining). The cultures
were also labeled with DAPI, a nuclear dye (FIG. 17B, blue ovals).
A phase contrast image of the microscopic field is shown in FIG.
17A. Note that the p75+ cells (FIG. 17B, solid arrow) do not
express SMA< whereas the SMA+ cells (FIG. 17B, open arrows) do
not express p75.
FIGS. 18 A and B demonstrate that individual neural cres cells can
generate neurons, glia and smooth muscle cells. The figures
illustrate three views of a clone derived from a single p75+ neural
crest founder cell, grown for two weeks in standard medium. A
neuron is identifiable in the clone by virtue of peripherin
expression (FIG. 18B, arrowhead) and long neurites (FIG. 18A). Glia
are identifiable by GFAP expression (FIG. 18C, orange staining,
open arrows), and a smooth muscle cell is identified by staining
with anti-SMA (FIG. 18C, green staining, closed arrow). Nuclei of
all cells have been labeled blue with DAPI (FIG. 18C).
FIGS. 19 A, B and C demonstrate that smooth muscle cell
differentiation is promoted by fetal bovine serum. Shown are three
views of a colony of neural crest cells grown in 5% fetal bovine
serum. These cells do not express p75-LNGFR under these conditions.
Cells visible by phase-contrast (FIG. 19A) express both SMA (FIG.
19B, red staining) and also desmin (FIG. 19C, green staining).
FIGS. 20 A and B demonstrate that neural crest-derived smooth
muscle cells express calponin. The culture is similar to that in
FIG. 19, except the cells were doubly-labeled with anti-SMA (FIG.
20B, red staining) and calponin (FIG. 20B, green staining). Cells
that co-express both markers stain orange due to blending of the
two colors (FIG. 20B).
DETAILED DESCRIPTION OF THE INVENTION
The invention is directed, in part, to the isolation and clonal
propagation of non-transformed mammalian neural crest stem cells
and to multipotent neural stem cells from other embryonic and adult
tissue. The invention also includes the production of neural crest
stem cell and multipotent neural stem cell derivatives including
progenitor and more differentiated cells of the neuronal and glial
lineages. The invention is illustrated using neural crest stem
cells isolated from the rat. The invention, however, encompasses
all mammalian neural crest stem cells and multipotent neural stem
cells and their derivatives and is not limited to neural crest stem
cells from the rat. Mammalian neural crest stem cells and
multipotent neural stem cells and their progeny can be isolated
from tissues from human and non-human primates, equines, canines,
felines, bovines, porcines, lagomorphs, etc.
The invention encompasses several important methodological
innovations: 1) the use of monoclonal antibodies to the
low-affinity Nerve Growth Factor Receptor (LNGFR) as a cell surface
marker to isolate and identify neural crest stem cells, a method
extensible to other neural stem cell populations as well; 2) the
development of cell culture substrates and medium compositions
which permit the clonal expansion of undifferentiated neural crest
cells; 3) the development of culture substrates and medium
compositions which permit the differentiation of mammalian neural
crest cells into their differentiated derivatives (including but
not restricted to peripheral neurons and glia) in clonal
culture.
The invention also provides neural crest stem cells and other
multipotent neural stem cells. It is important to understand that
such cells could not be identified as stem cells without the
development of the isolation and cell culture methodologies
summarized above. The identification of a neural stem cell requires
that several criteria be met: 1) that the cell be an
undifferentiated cell capable of generating one or more kinds of
differentiated derivatives; 2) that the cell have extensive
proliferative capacity; 3) that the cell be capable of self-renewal
or self-maintenance (Hall et al. (1989) Development 106:619; Potten
et al. (1990) Crypt. Development 110:1001). The concept of a stem
cell as obligatorily capable of "unlimited" self-renewal is
applicable only to regenerating tissues such as skin or intestine.
In the case of a developing embryo stem cells may have limited
self-renewal capacity but be stem cells nevertheless (Potten et al.
(1990) supra). The development of clonal culture methods permitted
the demonstration of criteria 1 and 2 herein. The development of
sub-clonal culture methods (i.e., the ability to clone single
neural stem cells, and then re-clone progeny cells derived from the
original founder cell) further permitted the demonstration herein
of criterion 3.
To appreciate the significance of this demonstration, consider an
alternative hypothesis for cells from the neural crest: individual
undifferentiated neural crest cells divide to generate both neurons
and glia (i.e., meet criteria 1 and 2 above), but the daughter
cells produced by these initial cell divisions are committed to
producing either neurons or glia, but not both. In this case, the
neural crest cell is a progenitor cell but not a stem cell, because
it does not have self-renewal capacity. If this were the case, then
upon sub-cloning of neural crest cell clones, the resulting
"secondary" clones could contain either neurons or glia, but not
both. This is not observed. Rather, most or all of the secondary
clones contain both neurons and glia, like their parent clones.
This experiment thus provides the first definitive evidence that
neural progenitor cells from any region of the nervous system have
stem cell properties. In no other set of published experiments have
these stringent criteria for stem cell properties been met, despite
claims that "stem cells" have been isolated or identified (Cattaneo
et al. (1991) Trends Neurosci. 14:338; Reynolds et al. (1992)
Science 255:1707) from the mammalian central nervous system. This
in part reflects imprecise use of the term "stem cell" and in part
the failure to perform adequate experimental tests to support the
existence of such cells.
As used herein, the term "non-transformed cells" means cells which
are able to grow in vitro without the need to immortalize the cells
by introduction of a virus or portions of a viral genome containing
an oncogene(s) which confers altered growth properties upon cells
by virtue of the expression of viral genes within the transformed
cells. These viral genes typically have been introduced into cells
by means of viral infection or by means of transfection with DNA
vectors containing isolated viral genes.
As used herein, the term "genetically-engineered cell" refers to a
cell into which a foreign (i.e., non-naturally occurring) nucleic
acid, e.g., DNA, has been introduced. The foreign nucleic acid may
be introduced by a variety of techniques, including, but not
limited to, calcium-phosphate-mediated transfection, DEAE-mediated
transfection, microinjection, retroviral transformation, protoplast
fusion and lipofection. The genetically-engineered cell may express
the foreign nucleic acid in either a transient or long-term manner.
In general, transient expression occurs when foreign DNA does not
stably integrate into the chromosomal DNA of the transfected cell.
In contrast, long-term expression of foreign DNA occurs when the
foreign DNA has been stably integrated into the chromosomal DNA of
the transfected cell.
As used herein, an "immortalized cell" means a cell which is
capable of growing indefinitely in culture due to the introduction
of an "immortalizing gene(s)" which confers altered growth
properties upon the cell by virtue of expression of the
immortalizing gene(s) within the genetically engineered cell.
Immortalizing genes can be introduced into cells by means of viral
infection or by means of transfection with vectors containing
isolated viral nucleic acid encoding one or more oncogenes. Viruses
or viral oncogenes are selected which allow for the immortalization
but preferably not the transformation of cells. Immortalized cells
preferably grow indefinitely in culture but do not cause tumors
when introduced into animals.
As used herein, the term "transformed cell" refers to a cell having
the properties of 1) the ability to grow indefinitely in culture
and 2) causing tumors upon introduction into animals.
"Transformation" refers to the generation of a transformed
cell.
As used herein, the term "feeder-cell independent culture" or
grammatical equivalents means the growth of cells in vitro in the
absence of a layer of different cells which generally are first
plated upon a culture dish to which cells from the tissue of
interest are added. The "feeder" cells provide a substratum for the
attachment of the cells from the tissue of interest and
additionally serve as a source of mitogens and survival factors.
The feeder-cell independent cultures herein utilize a chemically
defined substratum, for example fibronectin (FN) or poly-D-lysine
(PDL) and mitogens or survival factors are provided by
supplementation of the liquid culture medium with either purified
factors or crude extracts from other cells or tissues. Therefore,
in feeder-cell independent cultures, the cells in the culture dish
are primarily cells derived from the tissue of interest and do not
contain other cell types required to support the growth of the
cells derived from the tissue of interest.
As used herein, the term "clonal density" means a density
sufficiently low enough to result in the isolation of single,
non-impinging cells when plated in a culture dish, generally about
225 cells/100 mm culture dish.
As used herein, the term "neural crest stem cell" means a cell
derived from the neural crest which is characterized by having the
properties (1) of self-renewal and (2) asymmetrical division; that
is, one cell divides to produce two different daughter cells with
one being self (renewal) and the other being a cell having a more
restricted developmental potential, as compared to the parental
neural crest stem cell. The foregoing, however, is not to be
construed to mean that each cell division of a neural crest stem
cell gives rise to an asymmetrical division. It is possible that a
division of a neural crest stem cell can result only in
self-renewal, in the production of more developmentally restricted
progeny only, or in the production of a self-renewed stem cell and
a cell having restricted developmental potential.
As used herein, the term "multipotent neural stem cell" refers to a
cell having properties similar to that of a neural crest stem cell
but which is not necessarily derived from the neural crest. Rather,
as described hereinafter, such multipotent neural stem cells can be
derived from various other tissues including neural epithelial
tissue from the brain and/or spinal cord of the adult or embryonic
central nervous system or neural epithelial tissue which may be
present in tissues comprising the peripheral nervous system. In
addition, such multipotent neural stem cells may be derived from
other tissues such as lung, bone and the like utilizing the methods
disclosed herein. It is to be understood that such cells are not
limited to multipotent cells but may comprise a pluripotent cell
capable of regeneration and differentiation to different types of
neurons and glia, e.g., PNS and CNS neurons and glia or progenitors
thereof. In this regard, it should be noted that the neural crest
stem cells described herein are at least multipotent in that they
are capable, under the conditions described, of self-regeneration
and differentiation to some but not all types of neurons and glia
in vitro. Thus, a neural crest stem cell is a multipotent neural
stem cell derived from a specific tissue, i.e., the embryonic
neural tube.
In most embodiments, neural crest stem cells are further
characterized by a neural cell-specific surface marker. Such
surface markers in addition to being found on neural chest stem
cells may also be found on other multipotent neural stems derived
therefrom, e.g., glial and neuronal progenitor cells of the
peripheral nervous system (PNS) and central nervous system (CNS).
An example is the cell surface expression of a nerve growth factor
receptor on neural crest stem cells. In rat, humans and monkeys
this nerve growth factor receptor is the low-affinity nerve growth
factor receptor (LNGFR). Such stem cells may also be characterized
by the expression of nestin, an intracellular intermediate filament
protein. Neural crest stem cells may be further characterized by
the absence of markers associated with mature PNS neuronal or glial
cells. In the rat, such markers include sulfatide, glial fibrillary
acidic protein (GFAP) and myelin protein P.sub.o in PNS glial cells
and peripherin and neurofilament in PNS neuronal cells.
LNGFR is a receptor for nerve growth factor, a neurotrophic factor
shown to be responsible for neuronal survival in vivo. LNGFR is
found on several mammalian cell types including neural crest cells
and Schwann cells (glial cells of the PNS) as well as on the
surface of cells in the ventricular zone throughout the embryonic
central nervous systems. (See, e.g., Yan et al. (1988) J. Neurosci.
8:3481-3496 and Heuer, J. G. et al. (1980) Neuron 5:283-296 which
studied such cells in the rat and chick systems, respectively.)
Antibodies specific for LNGFR have been identified for LNGFR from
rat monoclonal antibodies 217c (Peng, W. W. et al. (1982) Science
215:1102-1104) and 192-Ig (Brockes, J. P. et al. (1977) Nature
266:364-366 and Chandler, C. E. et al. (1984) J. Biol. Chem.
259:6882-6889) and human (Ross, A. H. et al. (1984) Proc. Natl.
Acad. Sci. USA 81:6681-6685; Johnson, et al. (1986) Cell
47:545-554; Loy et al. (1990) J. Neurosci Res. 27:651-644). The
monoclonal antibody against human LNGFR has been reported to
cross-react with LNGFR from monkeys (Mufson, E. G. et al. (1991) J.
Comp. Neurol. 308:555-575). The DNA sequence has been determined
for rat and human LNGFR (Radeke, M. J. et al. (1987) Nature
325:593-597 and Chao, M. V. et al. (1986) Science 232:518-521,
respectively) and is highly conserved between rat and human.
Using the following techniques, monoclonal antibodies specific for
LNGFR from any desired mammalian species are generated by first
isolating the nucleic acid encoding the LNGFR protein. One protocol
for obtaining such nucleic acid sequences uses one or more nucleic
acid sequences from a region of the LNGFR gene which is highly
conserved between mammalian species, e.g., rat and human, as a
hybridization probe to screen a genomic library or a cDNA library
derived from mammalian tissue from the desired species (Sambrook,
J. et al. (1989) Cold Spring Harbor Laboratory Press. Molecular
Cloning: A Laboratory Manual, 2nd Ed., pp. 8.3-8.80, 9.47-9.58 and
11.45-11.55). The cloned LNGFR sequences are then used to express
the LNGFR protein or its extracellular (ligand binding) domain in
an expression host from which the LNGFR protein is purified.
Purification is performed using standard techniques such as
chromatography on gel filtration, ion exchange or affinity resins.
The purified LNGFR is then used to immunize an appropriate animal
(e.g., mouse, rat, rabbit, hamster) to produce polyclonal antisera
and to provide spleen cells for the generation of hybridoma cell
lines secreting monoclonal antibodies specific for LNGFR of the
desired species (Harlow, E. et al. (1988) Cold Spring Harbor
Laboratory Press, Antibodies: A Laboratory Manual, pp.
139-242).
A novel screening method can be used to detect the production of
antibody against LNGFR or any other surface marker which
characterizes a multipotent neural stem cell or progeny thereof.
The method can be practiced to detect animals producing polyclonal
antibodies against a particular antigen or to identify and select
hybridomas producing monoclonal antibodies against such antigens.
In this method, serum from an immunized animal or supernatant from
a hybridoma culture is contacted with a live neural cell which
displays a surface marker characteristic of a particular neural
cell line. Detection of whether binding has occurred or not is
readily determined by any number of known methods A particularly
preferred method is to use labeled antibody which is specific for
the immunoglobulins produced by the species which is immunized with
the particular antigen and which is a source for polyclonal serum
and spleen cells for hybridoma formation.
The live neural cell used in the foregoing antibody assay is
dependent upon the particular surface marker for which an antibody
is desired. In the examples, a monoclonal antibody for mouse LNGFR
was identified using a dissociated primary culture of Schwann
cells. In conjunction with the assay disclosed in the examples,
mouse fibroblasts acted as a negative control. However, primary
cultures of other cell lines can be used to detect monoclonal
antibodies to LNGFR. For example, forebrain cholinergic neurons or
sensory neurons can be used. In addition, a primary culture of
epithelial cells can be used as a negative control.
Other markers found on neural cells include Platelet Derived Growth
Factor Receptor (PDGFR), Fibroblast Growth Factor (FGF) and Stem
Cell Factor Receptor (SCFR). Cells useful for detecting monoclonal
antibodies to PDGFR and FGF include primary cultures of glial cells
or fibroblasts. Negative controls include cultures of epithileal
cells and neuroblastomas. SCFR is expressed on a subset of neuronal
cells. Primary cultures of melanocytes or melanoma cells can be
used to detect monoclonal antibodies to this receptor. Negative
controls include primary cultures of fibroblasts and glial
cells.
It is not always necessary to generate polyclonal or monoclonal
antibodies that are species specific. Monoclonal antibodies against
an antigenic determinant from one species may react against that
antigen from more than one species. For example, as stated above,
the antibody directed against the human LNGFR molecule also
recognizes LNGFR on monkey cells. When cross-reactive antibodies
are available, there is no need to generate antibodies which are
species specific using the methods described above.
Nestin, a second marker in the neural crest stem cell, is an
intermediate filament protein primarily located intracellularly,
which has been shown to be present in CNS neuroepithelial cells and
Schwann cells in the peripheral nervous system of rats (Friedman et
al. (1990) J. Comp. Neurol. 295:43-51). Monoclonal antibodies
specific for rat nestin have been isolated: Rat 401, (Hockfield, S.
et al. (1985) J. Neurosci. 5(12):3310-3328). A polyclonal rabbit
anti-nestin antisera has been reported which recognizes mouse
nestin (Reynolds, D. A. et al. (1992) Science 255:1707-1710). The
DNA sequences encoding the rat nestin gene have been cloned
(Lendahl, U. et al. (1990) Cell 60:585-595). These DNA sequences
are used to isolate nestin clones from other mammalian species.
These DNA sequences are then used to express the nestin protein and
monoclonal antibodies directed against various mammalian nestins
are generated as described above for LNGFR.
As used herein, the term "glial progenitor cell" refers to a cell
which is intermediate between the fully differentiated glial cell
and a precursor multipotent neural stem cell from which the fully
differentiated glial cell develops. In general, such glial
progenitor cells are derived according to the methods described
herein for isolating such cells from various tissues including
adult and embryonic CNS and PNS tissue as well as other tissues
which may potentially contain such progenitors.
As used herein, the term "PNS glial progenitor cell" means a cell
which has differentiated from a mammalian neural crest stem cell
which is committed to the PNS glial lineage and is a dividing cell
but does not yet express surface or intracellular markers found on
more differentiated, non-dividing PNS glial cells. Such progenitor
cells are preferably obtained from neural crest stem cells isolated
from the embryonic neural crest which have undergone further
differentiation. However, equivalent cells may be derived from
other tissue. When PNS glial progenitor cells are placed in
appropriate culture conditions they differentiate into PNS glia
expressing the appropriate differentiation markers, for example,
sulfatide and GFAP.
Sulfatide is a glycolipid molecule found on the surface of Schwann
cells and oligodendricytes in rats, mice, chickens and humans. The
expression of sulfatide on Schwann cells is dependent upon either
axonal contact or exposure to cyclic AMP or analogs thereof, such
as forskolin (Mirsky, R. et al. (1990) Development 109:105-116).
Monoclonal antibodies specific for sulfatide have been reported
(Sommer, I. et al. (1981) Dev. Biol. 83:311-327).
Glial fibrillary acidic protein (GFAP) is an intermediate filament
protein specifically expressed by astrocytes and glial cells of the
CNS and by Schwann cells, the glial cells of the PNS (Jessen, K. R.
et al. (1984) J. Neurocytology 13:923-934 and Fields, K. L. et al.
(1989) J. Neuroimmuno. 8:311-330). Monoclonal antibodies specific
for GFAP have been reported (Debus et al. (1983) Differentiation
25:193-203). Mouse and human GFAP genes have been cloned (Cowan, N.
J. et al. (1985) N.Y. Acad. Sci. 455:575-582 and Bongcamrudlowss,
D. et al. (1991) Cancer Res. 51:1553-1560, respectively). These DNA
sequences are used to isolate GFAP clones from other mammalian
species. These DNA sequences are then used to express the GFAP
protein and monoclonal antibodies directed against various
mammalian GFAPs are generated as described above for LNGFR.
As used herein, the term "factors permissive for PNS glial cell
differentiation" means compounds, such as, but not limited to,
protein or steroid molecules or substrates such as FN or PDL, which
permit at least neural crest stem cells to become restricted to the
PNS glial lineage. Such lineage-restricted progeny of neural crest
stem cells include glial progenitor cells, which are at least
bipotential, in that they can divide to give rise to self, as well
as, more mature non-dividing PNS glial cells.
As used herein, the term "neuronal progenitor cell" refers to a
cell which is intermediate between the fully differentiated
neuronal cell and a precursor multipotent neural stem cell from
which the fully differentiated neuronal cell develops. In general,
such neuronal progenitor cells are derived according to the methods
described herein for isolating such cells from various tissues
including adult and embryonic CNS and PNS tissue as well as other
tissues which may potentially contain such progenitors.
As used herein, the term "PNS neuronal progenitor cell" means a
cell which has differentiated from a mammalian neural crest stem
cell which is committed to one or more PNS neuronal lineages and is
a dividing cell but does not yet express surface or intracellular
markers found on more differentiated, non-dividing PNS neuronal
cells. Such progenitor cells are preferably obtained from neural
crest stem cells isolated from the embryonic neural crest which
have undergone further differentiation. However, equivalent cells
may be derived from other tissue. When PNS neuronal progenitor
cells are placed in appropriate culture conditions they
differentiate into mature PNS neurons expressing the appropriate
differentiation markers, for example, peripherin, neurofilament and
high-polysialic acid neural cell adhesion molecule (high
PSA-NCAM).
Peripherin, a 57 kDa intermediate filament protein, is expressed in
adult rodents primarily in peripheral neurons. More limited
expression of peripherin is found in some motoneurons of the spinal
cord and brain stem and a limited group of CNS neurons. Peripherin
is expressed in rat embryos primarily in neurons of peripheral
ganglia and in a subset of ventral and lateral motoneurons in the
spinal cord (Gorham, J. D. et al. (1990) Dev. Brain Res.
57:235-248). Antibodies specific for this marker have been
identified in the rat (Portier, M. et al. (1983/84) Dev. Neurosci.
6:335-344). The DNA sequences encoding the rat peripherin gene have
been cloned (Thompson, M. A. et al. (1989) Neuron 2:1043-1053).
These DNA sequences are used to isolate DNA sequences for the
peripherin gene in other mammals that are used to express the
protein and generate antibodies directed against other mammalian
peripherin proteins, as described above for LNGFR.
Neurofilaments are neuron-specific intermediate filament proteins.
Three neurofilament (NF) proteins have been reported: NF68, a 68 kD
protein also called NF-L (Light); NF160, a 160 kD protein also
called NF-M (Medium); NF200, a 200 kD protein also called NF-H
(Heavy). In general, there is coordinate expression of all three NF
proteins in neurons. The DNA sequences encoding the rat NF200 and
NF160 proteins have been cloned (Dautigny, A. et al. (1988)
Biochem. Biophys. Res. Commun. 154:1099-1106 and Napolitano, E. W.
et al. (1987) J. Neurosci. 7:2590-2599, respectively). All three NF
protein genes have been cloned in mice and humans. Mouse NF68
nucleic acid sequences were reported in Lewis, S. A. et al. (1985)
J. Cell Biol. 100:843-850. Mouse NF160 nucleic acid sequences were
reported in Levy, E. et al. (1987) Eur. J. Biochem. 166:71-77.
Mouse NF200 nucleic acid sequences were reported in Shneidman, P.
S. et al. (1988) Mol. Brain Res. 4:217-231. In humans, nucleic acid
sequences were reported for: NF68, Julien, J.-P. et al. (1987)
Biochem. Biophys. Acta. 909:10-20; NF160, Myers, M. W. et al.
(1987) EMBO J. 6:1617-1626; NF200, Lee, J. F. et al. (1988) EMBO J.
7:1947-1955. These DNA sequences are used to produce the protein
for the production of antibodies or to isolate other mammalian NF
genes and the proteins expressed and antibodies generated for any
desired species, as described above for LNGFR. As used herein, the
term "NF.sup.+ " means expression of one or more of the three NF
proteins.
As used herein, the term "factors permissive for PNS neuronal cell
differentiation" means compounds, such as, but not limited to,
protein or steroid molecules or substrates such as FN or PDL, which
permit at least a neural crest stem cell to become restricted to
the PNS neuronal lineage. Such lineage-restricted progeny of neural
crest stem cells include PNS neuronal progenitor cells, which are
at least bipotential, in that they can divide to give rise to self,
as well as, more mature, non-dividing PNS neurons.
As indicated in the examples, when neural stem cells are contacted
with certain factors permissive for neuronal and glial cell
differentiation, such cells differentiated into neurons, glia and a
subpopulation referred to as "O" cells. As disclosed in Example 10,
these O cells are, in fact, smooth muscle cells. Thus, at least
some of the factors which are permissive for differentiation to
neuronal and/or glial cells are also permissive for the
differentiation of neural stem cells to smooth muscle cells.
However, as also indicated in Example 10, there are factors which
are instructive for smooth muscle cell differentiation. In this
regard, the term "instructive factor" or grammatical equivalents
refers to one or more factors which are capable of causing the
differentiation of neural stem cells primarily to a single lineage,
e.g., glial, neuronal or smooth muscle cell. Thus, a factor which
is instructive for smooth muscle cell differentiation is one which
causes differentiation of neural stem cells to smooth muscle cells
at the expense of the differentiation of such stem cells into other
lineages such as glial or neuronal cells. As indicated in Example
10, mammalian serum contains one or more factors which are
instructive factors for the production of smooth muscle cells.
Having identified that mammalian serum contains one or more
instructive factors for smooth muscle cell differentiation, such
instructive factors can be identified by fractionating mammalian
serum and adding back one or more such fractions to a neural stem
cell culture to identify one or more fractions containing
instructive factors for smooth muscle cell differentiation.
Positive fractions can then be further fractionated and reassayed
until the one or more components required for instructive
differentiation to smooth muscle cells are identified.
Mammalian neural crest stem cell compositions are provided which
serve as a source for neural crest cell derivatives such as
neuronal and glial progenitors of the PNS which in turn are a
source of PNS neurons and glia. Methods are provided for the
isolation and clonal culture of neural crest stem cells, in the
absence of feeder cells. In the examples provided, these methods
utilize a chemically defined medium which is supplemented with
chick embryo extract as a source of mitogens and survival factors.
Factors present in the extract of chicken embryos allow the growth
and self renewal of rat neural crest stem cells. However, media
used to isolate and propagate rat neural crest stem cells can be
used to isolate and propagate neural crest stem cells from other
mammalian species, such as human and non-human primates, equines,
felines, canines, bovines, porcines, lagomorphs, etc.
Culture conditions provided herein allow the isolation self-renewal
and differentiation of mammalian neural crest stem cells and their
progeny. These culture conditions may be modified to provide a
means of detecting and evaluating growth factors relevant to
mammalian neural crest stem cell self-renewal and the
differentiation of the stem cell and its progeny. These
modifications include, but are not limited to, changes in the
composition of the culture medium and/or the substrate and in the
specific markers used to identify either the neural crest stem cell
or their differentiated derivatives.
Culture conditions are provided which allow the differentiation of
manunalian neural crest stem cells into the PNS neuronal and glial
lineages in the absence of feeder cell layers. In addition to
liquid culture media, these culture conditions utilize a substratum
comprising fibronectin alone or in combination with poly-D-lysine.
In the examples provided, human fibronectin is utilized for the
culturing of rat neural crest stem cells and their progeny. Human
fibronectin can be used for the culturing of neural crest stem
cells isolated from avian species as well as from any mammal, as
the function of the fibronectin protein is highly conserved among
different species. Cells of many species have fibronectin receptors
which recognize and bind to human fibronectin.
In order to isolate the subject neural crest stem cells, it is
necessary to separate the stem cell from other cells in the embryo.
Initially, neural crest cells are obtained from mammalian
embryos.
For isolation of neural crest cells from mammalian embryos, the
region containing the caudal-most 10 somites are dissected from
early embryos (equivalent to gestational day 10.5 day in the rat).
These trunk sections are transferred in a balanced salt solution to
chilled depression slides, typically at 4.degree. C., and treated
with collagenase in an appropriate buffer solution such as Howard's
Ringer's solution. After the neural tubes are free of somites and
notochords, they are plated onto fibronectin (FN)-coated culture
dishes to allow the neural crest cells to migrate from the neural
tube. Twenty-four hours later, following removal of the tubes with
a sharpened tungsten needle, the crest cells are removed from the
FN-coated plate by treatment with a Trypsin solution, typically at
0.05%. The suspension of detached cells is then collected by
centrifugation and plated at an appropriate density, generally 225
cells/100 mm dish in an appropriate chemically defined medium. This
medium is preferentially free of serum and contains components
which permit the growth and self-renewal of neural crest stem
cells. The culture dishes are coated with an appropriate
substratum, typically a combination of FN and poly-D-lysine
(PDL).
Procedures for the identification of neural crest stem cells
include incubating cultures of crest cells for a short period of
time, generally 20 minutes, at room temperature, generally about
25.degree. C., with saturating levels of antibodies specific for a
particular marker, e.g., LNGFR. Excess antibody is removed by
rinsing the plate with an appropriate medium, typically L15 medium
(Gibco) supplemented with fresh vitamin mix and bovine serum
albumin (L-15 Air). The cultures are then incubated at room
temperature with a fluorochrome labelled secondary antibody,
typically Phycoerythrin R-conjugated secondary antibody (TAGO) at
an appropriate dilution for about 20 minutes. Excess secondary
antibodies are then removed using an appropriate medium, such as
L-15 Air. The plates are then covered with the chemically defined
growth medium and examined with a fluorescence microscope.
Individual LNGFR.sup.+ clones are isolated by fluorescence
activated cell sorting (FACS) or, more typically, by marking the
plate under the identified clone. The markings are typically made
to a diameter of 3-4 mm, which generally allows for the unambiguous
identification of the progeny of the founder cell at any time
during an experiment. If desired, individual LNGFR.sup.+ clones are
removed from the original plate by trypsinization with the use of
cloning cylinders.
Procedures for permitting the differentiation of stem cells include
the culturing of isolated stem cells in a medium permissive for
differentiation to a desired lineage, such as Schwann cell
differentiation (SCD) medium. Other procedures include growth of
isolated stem cells on substrates capable of permitting
differentiation, such as FN or FN and PDL.
Procedures for the serial subcloning of stem cells and their
derivatives include the trypsinization of individual clones, as
described above, followed by replating the clone on a desired
substrate and culturing in a desired medium, such as a chemically
defined medium suitable for maintenance of stem cells or SCD medium
permissive for the differentiation of said neural crest stem cells.
Crest cells may be identified following serial subcloning by
live-cell labeling with an antibody directed against LNGFR, as
described above.
The methods described herein provide the basis of functional assays
which allow for the identification and production of cellular
compositions of mammalian cells which have properties
characteristic of neural crest stem cells, glial or neuronal
progenitor cells or multipotent stem cell precursor of such
progenitor cells. In order to isolate such cells from tissues other
than embryonic neural tubes, it is necessary to separate the
progenitor and/or multipotent stem cells from other cells in the
tissue. The methods presented in the examples for the isolation of
neural crest stem cells from neural tubes can be readily adapted
for other tissues by one skilled in the art. First, a single cell
suspension is made from the tissue; the method used to make this
suspension will vary depending on the tissue utilized. For example,
some tissues require mechanical disruption of the tissue while
other tissues require digestion with proteolytic enzymes alone or
in combination with mechanical disruption in order to create the
single cell suspension. Tissues such as blood already exists as a
single cell suspension and no further treatment is required to
generate a suspension, although hypotonic lysis of red blood cells
may be desirable. Once the single cell suspension is generated it
may be enriched for cells expressing LNGFR or other neural
cell-specific markers on their surface. One protocol for the
enrichment for LNGFR.sup.+ cells is by incubating the cell
suspension with antibodies specific for LNGFR and isolating the
LNGFR.sup.+ cells. Enrichment for cells expressing a neural
cell-specific surface marker is particularly desirable when these
cells represent a small percentage (less than 5%) of the starting
population. The isolation of cells which have complexed with an
antibody for a neural cell-specific surface marker such as is
carried out using any physical method for isolating
antibody-labeled cells. Such methods include fluorescent-activated
cell sorting in which case the cells, in general, are further
labeled with a fluorescent secondary antibody that binds the
anti-LNGFR antibody, e.g., mouse anti-LNGFR and fluorescein label
goat anti-mouse IgG; panning in which case the antibody-labeled
cells are incubated on a tissue-culture plate coated with a
secondary antibody; Avidin-sepharose chromatography in which the
anti-LNGFR antibody is biotinylated prior to incubation with the
cell suspension so that the complexed cells can be recovered on an
affinity matrix containing avidin (i.e., where the antibody is an
antibody conjugate with one of the members of a binding pair); or
by use of magnetic beads coated with an appropriate anti-antibody
so that the labeled LNGFR-expressing cells can be separated from
the unlabeled cells with the use of a magnet. All of the foregoing
cell isolation procedures are standard published procedures that
have been used previously with other antibodies and other
cells.
The use of antibodies specific for neural stem cell-specific
surface markers results in the isolation of multipotent neural stem
cells from tissues other than embryonic neural tubes. For example,
as previously indicated, LNGFR is expressed in cells of the
ventricular zone throughout the embryonic central nervous system of
the rat and chick. This implies that other mammalian species have a
similar pattern of LNGFR expression and studies in human with
monoclonal antibodies against the human LNGFR (Loy, et al. (1990)
J. Neurosci. Res. 27:651-654) are consistent with this expectation.
Since cells from the ventricular zone (Cattaneo et al. (1991)
Trends Neurosci. 14:338-340; Reynolds et al. (1992) Science
255:1707-1710) are likely to be stem cells (Hall et al. (1989)
Development 106:619-633; Potter et al. (1990) Development
110:1001-1020) antibodies to neural cell-specific surface markers
should prove useful in isolating multipotent neural stem cells from
the central and peripheral nervous systems and from other tissue
sources.
Alternatively, or in conjunction with the above immuno-isolation
step, the cells are plated at clonal density, generally 225
cells/100 mm dish, in an appropriate chemically defined medium on a
suitable substrate as described in the examples for isolation of
rat neural crest stem cells. The presence of neural crest-like stem
cells (e.g., a multipotent neural stem cell) is confirmed by
demonstrating that a single cell can both self-renew and
differentiate to members of at least the PNS neuronal and glial
lineages utilizing the culture conditions described herein. Other
types of multipotent neural stem cells are identified by
differentiation to other cell type such as CNS neural or glial
cells or their progenitors. Depending upon the source of the tissue
used in the foregoing methods, multipotent neural stem cells may
not be obtained. Rather, further differentiated cell types such as
glial and neuronal progenitor cells may be obtained.
Transplantation assay systems described herein provide the basis of
functional assays which allow for the identification of mammalian
cells which have properties characteristic of neural crest stem
cells, multipotent neural stem cells and/or neuronal or glial
progenitor cells. Cells of interest, identified by either the in
vivo or in vitro assays described above, are transplanted into
mammalian hosts using standard surgical procedures. The
transplanted cells and their progeny are distinguished from the
host cells by the presence of species specific antigens or by the
expression of an introduced marker gene. The transplanted cells and
their progeny are also stained for markers of mature neurons and
glia in order to examine the developmental potential of the
transplanted cells. This transplantation assay provides a means to
identify neural crest stem cells by their functional properties in
addition to the in vitro culture assays described above.
Additionally, the transplantation of cells having characteristics
of multipotent neural stem cells, neural crest stem cells or
progenitors of neuronal or glial cells provides a means to
investigate the therapeutic potential of these cells for
neurological disorders of the PNS and CNS in animal models.
Examples of PNS disorders in mice include the trembler and shiverer
strains. The trembler mutation is thought to involve a defect in
the structural gene for myelin basic protein (MBP). This mutation
maps to the same region of chromosome 11 as does the MBP gene. This
mutation results in the defective myelination of axons in the PNS.
An analogous disorder is seen in humans, Charcot-Marie-Tooth
syndrome, which results in progressive neuropathic muscular
atrophy.
The shiverer mutation in mice results in a severe myelin deficiency
throughout the CNS and a moderate hypo-myelination in the PNS.
Severe shivering episodes are seen 12 days after birth. An
analogous disorder is seen in humans, Guillaum-Barre disease, which
is characterized by an acute febrile polyneuritis.
Cells having characteristics of multipotent neural stem cells,
neural crest stem cells or neuronal or glial progenitors of the PNS
or CNS (identified by either in vitro or in vivo assays) are
introduced into a mammal exhibiting a neurological disorder to
examine the therapeutic potential of these cells. These cells are
preferably isolated from a mammal having similar MHC genotypes or
the host mammal is immunosuppressed using drugs such as cyclosporin
A. The cells are injected into an area containing various
peripheral nerves known to be effected in a particular mammal or
into the spinal cord or brain for mammals which show involvement of
the CNS. The cells are injected at a range of concentrations to
determine the optimal concentration into the desired site.
Alternatively, the cells are introduced in a plasma clot or
collagen gel to prevent rapid dispersal of cells from the site of
injection. The effect of this treatment on the neurological status
of the model animal is noted. Desired therapeutic effects in the
above mutant mice include the reduction or cessation of seizures or
improved movement of lower motor extremities.
There is strong interest in identifying the multipotent neural stem
cells such as the neural crest stem cell and defining culture
conditions which allow the clonal propagation and differentiation
of said stem cells. Having possession of a multipotent neural stem
cell or a neural crest stem cell allows for identification of
growth factors associated with self regeneration. In addition,
there may be as yet undiscovered growth factors associated with (1)
with the early steps of restriction of the stem cell to a
particular lineage; (2) the prevention of such restriction; and (3)
the negative control of the proliferation of the stem cell or its
derivatives.
The multipotent neural stem cell, neural crest stem cell, progeny
thereof or immortalized cell lines derived therefrom are useful to:
(1) detect and evaluate growth factors relevant to stem cell
regeneration; (2) detect and isolate ligands, such as growth
factors or drugs, which bind to receptors expressed on the surface
of such cells or their differentiated progeny (e.g., Glial Growth
Factor (GGF), Heregulin and Neu Differentiation Factor (NDF)); (3)
provide a source of cells which express or secrete growth factors
specific to multipotent neural stem cells; (4) detect and evaluate
other growth factors relevant to differentiation of stem cell
derivatives, such as neurons and glia; (5) produce various neural
stem cell derivatives, including both the progenitors and mature
cells of a given lineage and (6) provide a source of cells useful
for treating neurological diseases of the PNS and CNS in model
animal systems and in humans. The culture conditions used herein
allow for the growth and differentiation of stem cells in vitro and
provide a functional assay whereby mammalian tissues can be assayed
for the presence of cells having the characteristics of neural stem
cells. The transplantation assay described herein also provides a
functional assay whereby mammalian neural stem cells may be
identified.
As indicated in the examples, neural crest stem cells have been
passaged for at least six-ten generations in culture. Although it
may be unnecessary to immortalize those or other multipotent neural
stem cell lines or progenitor cell lines obtained by the methods
described herein, once a cell line has been obtained it may be
immortalized to yield a continuously growing cell line useful for
screening trophic or differentiation factors or for developing
experimental transplantation therapies in animals. Such
immortalization can be obtained in multipotent neural stem cells or
progenitors of glial and neuronal cells by genetic modification of
such cells to introduce an immortalizing gene.
Examples of immortalizing genes include: (1) nuclear oncogenes such
as v-myc, N-myc, T antigen and Ewing's sarcoma oncogene
(Fredericksen et al. (1988) Neuron 1:439-448; Bartlett, P. et al.
(1988) Proc. Natl. Acad. Sci. USA 85:3255-3259, and Snyder, E. Y.
et al. (1992) Cell 68:33-51), (2) cytoplasmic oncogenes such as
bcr-abl and neurofibromin (Solomon, E. et al. (1991) Science
254:1153-1160), (3) membrane oncogenes such as neu and ret
(Aaronson, A. S. A. (1991) Science 254:1153-1161), (4) tumor
suppressor genes such as mutant p53 and mutant Rb (retinoblastoma)
(Weinberg, R. A. (1991) Science 254:1138-1146), and (5) other
immortalizing genes such as Notch dominant negative (Coffman, C. R.
et al. (1993) Cell 23:659-671). Particularly preferred oncogenes
include v-myc and the SV40 T antigen.
Foreign (heterologous) nucleic acid may be introduced or
transfected into multipotent neural stem cells or their progeny. A
multipotent neural stem cell or its progeny which harbors foreign
DNA is said to be a genetically-engineered cell. The foreign DNA
may be introduced using a variety of techniques. In a preferred
embodiment, foreign DNA is introduced into multipotent neural stem
cells using the technique of retroviral transfection. Recombinant
retroviruses harboring the gene(s) of interest are used to
introduce marker genes, such as the E. coli .beta.-galactosidase
(lacZ) gene, or oncogenes. The recombinant retroviruses are
produced in packaging cell lines to produce culture supernatants
having a high titer of virus particles (generally 10.sup.5 to
10.sup.6 pfu/ml). The recombinant viral particles are used to
infect cultures of the neural stem cells or their progeny by
incubating the cell cultures with medium containing the viral
particles and 8 .mu.g/ml polybrene for three hours. Following
retroviral infection, the cells are rinsed and cultured in standard
medium. The infected cells are then analyzed for the uptake and
expression of the foreign DNA. The cells may be subjected to
selective conditions which select for cells that have taken up and
expressed a selectable marker gene.
In another preferred embodiment, the foreign DNA is introduced
using the technique of calcium-phosphate-mediated transfection. A
calcium-phosphate precipitate containing DNA encoding the gene(s)
of interest is prepared using the technique of Wigler et al. (1979)
Proc. Natl. Acad. Sci. USA 76:1373-1376. Cultures of the neural
stem cells or their progeny are established in tissue culture
dishes. Twenty four hours after plating the cells, the calcium
phosphate precipitate containing approximately 20 .mu.g/ml of the
foreign DNA is added. The cells are incubated at room temperature
for 20 minutes. Tissue culture medium containing 30 .mu.M
chloroquine is added and the cells are incubated overnight at
37.degree. C. Following transfection, the cells are analyzed for
the uptake and expression of the foreign DNA. The cells may be
subjected to selection conditions which select for cells that have
taken up and expressed a selectable marker gene.
The following is presented by way of example and is not to be
construed as a limitation on the scope of the invention. Further,
all references referred to herein are expressly incorporated by
reference.
EXAMPLE 1
Preparation of Neural Crest Cells
For a given preparation 5-10 timed pregnant female Sprague-Dawley
rats (Simonson Laboratories, Gilroy, Calif.) were killed by
CO.sub.2 asphyxiation. Embryos were removed and placed into Hank's
Balanced Salt Solution (HBSS) (Gibco, Grand Island, N.Y.) at
4.degree. C. for 2-4 hours. Under a dissecting microscope, at room
temperature, a block of tissue from a region corresponding to
approximately the caudal most 10 somites was dissected from each
embryo using an L-shaped electrolytically sharpened tungsten
needle. Trunk sections were transferred in HBSS into one well of a
3 well depression slide that had been chilled to 4.degree. C. Trunk
sections were treated with collagenase (152 units/mg) (Worthington
Biochemical, Freehold, N. J.) made to a concentration of 0.75 mg/ml
in Howard's Ringer's solution (per 1 liter of dH.sub.2 O: NaCl 7.2
g; CaCl.sub.2 0.17 g; KCl 0.37 g) and sterilized, by passage
through a 0.22 .mu.m filter prior to use. The collagenase solution
was exchanged at least 3 times and with each exchange the trunk
sections were vigorously triturated by passage through a pasteur
pipet. After incubation at 37.degree. C. for 20 minutes in
humidified CO.sub.2 atmosphere, the trunk sections were triturated
very gently until most of the neural tubes were free and clean of
somites and notochords. The collagenase solution was quenched by
repeated exchanges with cold complete medium (described below). The
neural tubes were plated onto fibronectin-coated (substrate
preparation is described below) 60 mm tissue culture dishes
(Corning, Corning, N.Y.) that had been rinsed with complete medium.
After a 30 minute incubation to allow the neural tubes to attach,
dishes were flooded with 5 ml of medium. After a 24 hour culture
period, using an L-shaped electrolytically sharpened tungsten
needle and an inverted phase contrast microscope equipped with a
4.times. objective lens, each neural tube was carefully scraped
away from the neural crest cells that had migrated onto the
substrate. Crest cells were removed by a 2 minute 37.degree. C.
treatment with 0.05% Trypsin solution (Gibco). The cells were
centrifuged for 4 minutes at 2000 r.p.m. and the pellet was
resuspended into 1 ml of fresh complete medium. Typically the cells
were plated at a density of 225 cells/100 mm dish.
Substrate Preparation
A. Fibronectin (FN) Substrate
Tissue culture dishes were coated with human plasma fibronectin
(New York Blood Center, New York, N.Y.) in the following way.
Lyophilized fibronectin was resuspended in sterile distilled water
(dH.sub.2 O) to a concentration of 10 mg/ml and stored at
-80.degree. C. until used. The fibronectin stock was diluted to a
concentration of 250 mg/ml in Dulbecco's phosphate buffered saline
(D-PBS) (Gibco). The fibronectin solution was then applied to
tissue culture dishes and immediately withdrawn.
B. Poly-D-Lysine (PDL) and FN Substrate
Sterile poly-D-Lysine (PDL) was dissolved in dH.sub.2 O to as
concentration of 0.5 mg/ml. The PDL solution was applied to tissue
culture plates and immediately withdrawn. The plates were allowed
to dry at room temperature, rinsed with 5 ml of dH.sub.2 and
allowed to dry again. Fibronectin was then applied, as described
above, over the PDL.
EXAMPLE 2
Development of a Defined Medium for the Growth of Rat Neural Crest
Stem Cells
A serum-free, chemically defined basal medium was developed based
on the formulations of several existing defined media. This basal
medium consists of L15-CO.sub.2 formulated as described by Hawrot,
E. et al. (1979) Methods in Enzymology 58:574-583 supplemented with
additives described by Bottenstein, J. E. et al. (1979) Proc. Natl.
Acad. Sci. USA 76:514-517 and further supplemented with the
additives described by Sieber-Blum, M. et al. (1985) Exp. Cell Res.
158:267-272. The final recipe is given here: to L15-CO.sub.2 add,
100 .mu.g/ml transferrin (Calbiochem, San Diego, Calif.), 5
.mu.g/ml insulin (Sigma, St. Louis, Mo.), 16 .mu.g/ml putrescine
(Sigma), 20 nM progesterone (Sigma), 30 nM selenious acid (Sigma),
1 mg/ml bovine serum albumin, crystallized (Gibco), 39 pg/ml
dexamethasone (Sigma), 35 ng/ml retinoic acid (Sigma), 5 .mu.g/ml
.alpha.-d, 1-tocopherol (Sigma), 63 .mu.g/ml p-hydroxybuyrate
(Sigma), 25 ng/ml cobalt chloride (Sigma), 1 .mu.g/ml biotin
(Sigma), 10 ng/ml oleic acid (Sigma), 3.6 mg/ml glycerol, 100 ng/ml
.alpha.-melanocyte stimulating hormone (Sigma), 10 ng/ml
prostaglandin El (Sigma), 67.5 ng/ml triiodothyronine (Aldrich
Chemical Company, Milwaukee, Wisc.), 100 ng/ml epidermal growth
factor (Upstate Biotechnology, Inc., Lake Placid, N.Y.), 4 ng/ml
bFGF (UBI), and 20 ng/ml 2.55 NGF (UBI).
To allow the growth and regeneration of neural crest stem cells in
feeder cell-independent cultures, it was necessary to supplement
the basal medium with 10% chick embryo extract (CEE). This
supplemented medium is termed complete medium.
CEE is prepared as follows: chicken eggs were incubated for 11 days
at 38.degree. C. in a humidified atmosphere. Eggs were washed and
the embryos were removed, and placed into a petri dish containing
sterile Minimal Essential Medium (MEM with Glutamine and Earle's
salts) (Gibco) at 4.degree. C. Approximately 10 embryos each were
macerated by passage through a 30 ml syringe into a 50 ml test tube
(Corning). This typically produced 25 ml of volume. To each 25 ml
was added 25 ml of MEM. The tubes were rocked at 4.degree. C. for 1
hour. Sterile hyaluronidase (1 mg/25 g of embryo) (Sigma) was added
and the mixture was centrifuged for 6 hours at 30,000 g. The
supernatant was collected, passed first through a 0.45 .mu.m
filter, then through a 0.22 .mu.m filter and stored at -80.degree.
C. until used.
At the low cell densities necessary for survival and proliferation
of individual neural crest cells, either fetal calf serum (FCS, JR
Scientific) or CEE was required, in addition to the basal medium,
for clone formation. When FCS was used to supplement the medium, it
was heat inactivated by treatment at 55.degree. C. for 30 minutes.
FCS was stored at -20.degree. C. and passed through a 0.22 .mu.m
filter prior to use.
CEE is preferred as a supplement, as in the presence of FCS, most
of the cells derived from the neural crest exhibit a flattened,
fibroblastic morphology and expression of LNGFR is extinguished. In
the absence of both FCS and CEE, clone formation from neural crest
cells was greatly attenuated.
EXAMPLE 3
Isolation and Cloning of Multipotent Rat Neural Crest Cells
A. Identification of Antibody Markers Expressed by Neural Crest
Cells
In order to identify and isolate rat neural crest cells, it was
necessary to identify antibody markers that could be used to
recognize these cells. When E10.5 neural tubes were explanted onto
a fibronectin (FN) substratum, many of the neural crest cells that
emigrated from the neural tubes over the next 24 hours expressed
the low-affinity NGF receptor (LNGFR), recognized by monoclonal
antibodies 192-Ig and 217c. The outgrowth of neural crest cells
from the dorsal side of the explanted neural tube following 24
hours growth in culture is shown in FIG. 1, panel A. FIG. 1, panel
B shows the expression of LNGFR (green florescence) and nest in
(red fluorescence) in neural crest cells.
Neural crest cells were labeled with antibodies as follows: For
cell surface antigens, such as LNGFR, it was possible to label the
living cells in culture. The cultures were incubated with primary
antibody solution for 20 minutes at room temperature. The cultures
were washed twice with L15 medium (Gibco) supplemented with 1:1:2,
fresh vitamin mix (FVM) (Hawrot, E. et al. (1979), ibid), and 1
mg/ml bovine serum albumin (L15 Air). The cultures were then
incubated for 20 minutes at room temperature with Phycoerythrin R
conjugated secondary antibody (TAGO) at a dilution of 1:200 in L-15
Air. The cultures were then rinsed twice with L-15 Air and placed
back in their original medium and examined with a fluorescence
microscope. Rabbit anti-LNGFR antiserum (Weskamp, G. et al. (1991)
Neuron 6:649-663) was a kind gift of Gisela Weskamp, University of
California, San Francisco and was used at a 1:1000 dilution.
Monoclonal anti-NCAM antibody 5A5 (Dodd, J. et al. (1988) Neuron
1:105-116) and monoclonal anti-sulfatide antibody O.sub.4 (Sommer,
I. et al. (1981) Dev. Biol. 83:311-327) were obtained as hybridoma
cells from the Developmental Studies Hybridoma Bank (Johns Hopkins
University, Baltimore, Md.) and prepared as described by the
provider.
In order to label cells with antibodies directed against
intracellular proteins, it was necessary to fix and permeabilize
the cells prior to labeling. For most of the immunocytochemistry,
formaldehyde fixation was done. Formaldehyde solution 37% was
diluted 1:10 into S-MEM with 1 mM HEPES buffer (Gibco). Culture
were treated for 10 minutes at room temperature with the 3.7%
formaldehyde solution and then rinsed 3 times with D-PBS
(Gibco).
For some intermediate filament proteins (NF and GFAP) formaldehyde
fixation was not possible. Cultures were fixed by treatment with a
solution of 95% ethanol and 5% glacial acetic acid at -20.degree.
C. for 20 minutes.
For the staining of cytoplasmic antigens, fixed cells were first
treated with a blocking solution comprising D-PBS, 0.1% Tween-20
(Bio-Rad Laboratories, Richmond, Calif.) and 10% heat inactivated
normal goat serum (NGS) for 15 minutes at room temperature. Primary
antibodies were diluted with a solution of D-PBS, 0.1% Tween-20 and
5% NGS. The fixed cells were incubated overnight at 4.degree. C. in
primary antibody solution then rinsed twice with DPBS, 0.05%
Tween-20. Fluorescent secondary antibodies were diluted with D-PBS,
1% NGS and applied to cells for 1 hour at room temperature. The
cells were rinsed twice with D-PBS, 0.05% Tween-20. To prevent
photobleaching, a solution of 8 mg/ml N-propyl gallate in glycerol
was placed over the stained cells prior to fluorescence
microscopy.
Mouse monoclonal anti-GFAP, G-A-5 (Debus et al. (1983)
Differentiation 25:193-203) was purchased from Sigma and used at a
1:100 dilution. Mouse monoclonal anti-NF200, SMI39 was purchased
from Sternberger Monoclonals Inc., Baltimore, Md. and used at a
1:100 dilution. SMI39 reactivity is equivalent to the 06-53
monoclonal antibody described by Sternberger, L. A. et al. (1983)
Proc. Natl. Acad. Sci. USA 80:6126-6130. Purified rabbit antibodies
to peripherin (preparation 199-6) was obtained from Dr. Linda
Parysek, University of Cincinnati, Ohio and was used at a dilution
of 1:500.
Flow-cytometric analysis indicated that greater than 70% of the
neural crest cells show some LNGFR immunoreactivity (FIG. 1, panel
D). Approximately 25% of the neural crest cells expressed high
levels of LNGFR. In some experiments, neural crest cells expressing
high levels of LNGFR were further purified by labeling with 192-Ig
(anti-LNGFR) and fluorescence-activated cell sorting (FACS). For
single cell analysis, however, it proved more convenient to plate
the bulk neural crest cell population at clonal density, and then
subsequently identify LNGFR-positive cells by live cell-labeling
with 192-Ig.
Most or all of the neural crest cells also expressed nestin, an
intermediate filament protein found in CNS neuroepithelial cells.
An individual neural crest cell co-expressing both nestin and LNGFR
is shown in FIG. 2, panels A-C. Panel A shows the individual neural
crest cell in phase contrast. Panels B and C show this cell
following staining with both anti-LNGFR (panel B) and anti-nestin
(panel C). FIG. 2, panels D-F show that the clonal progeny of this
nestin.sup.+, LNGFR.sup.+ neural crest cell also co-express nestin
and LNGFR.
B. Cloning of Multipotent Neural Crest Cells
To define the developmental potential of individual neural crest
cells, conditions were established that permit the growth of these
cells in clonal culture. FIG. 3 provides a flow chart depicting the
following cell cloning experiments. In FIG. 3, plating medium
refers to the complete medium, described above and differentiation
medium refers to SCD medium, described below. Using an FCS-free,
CEE-containing medium (complete or plating medium), single neural
crest cells (FIG. 4, panel A, phase contrast and panel B, LNGFR
staining) were plated on a FN/PDL substratum and allowed to
proliferate and differentiate. After 9-14 days, many of the clones
founded by single neural crest cells were large and contained cells
with a neuronal morphology (FIG. 4, panel C, phase contrast).
Quantification indicated that >60% of the clones contained a
mixture of neuronal and non-neuronal cells (see below). These
neuronal cells could be labeled by antibodies to pan-neuronal
markers such as neurofilament (FIG. 4, panel E, anti-NF160
staining) and high-polysialyic acid (PSA) NCAM (FIG. 4, panel D,
anti-NCAM staining), as well as by an antibody to peripherin, an
intermediate filament protein that is preferentially expressed by
peripheral nervous system (PNS) neurons (FIG. 4, panel F).
Importantly, these neurons did not express either nestin or LNGFR,
indicating that they have lost the two markers that characterize
the undifferentiated neural crest cell.
The neuron-containing clones also contained non-neuronal cells.
These cells continued to express LNGFR and nestin, in contrast to
the neurons, and displayed an elongated morphology characteristic
of Schwann cells. While immature Schwann cells are known to express
both LNGFR and nestin, these markers are insufficient to identify
Schwann cells in this system since they are expressed by the neural
crest precursor cell as well. Expression of more definitive Schwann
cell markers was elicited by transferring the cells into a medium
known to enhance Schwann cell differentiation. This medium, called
Schwann cell differentiation (SCD) medium, contained both 10% FCS
and 5 .mu.M forskolin, an activator of adenylate cyclase.
FIG. 5 shows the expression of a Schwann cell phenotype by neural
crest-derived glia. Clones plated initially on FN were allowed to
grow for a week in complete medium, then transferred into SCD
medium and allowed to grow for another 1-2 weeks prior to fixation
and immunocytochemistry. Cells of two morphologies, one elongated
and the other flattened can be seen in phase contrast (Panels A and
D). To demonstrate concordant expression of three markers, LNGFR,
0.sub.4 and GFAP, two different double-labeling experiments were
performed. Living cells were surface-labeled with monoclonal
anti-LNGFR 192IgG (Panel B) and monoclonal 0.sub.4 IgM (Panel C)
and postfixed. In parallel, other cells from the same clone were
first surface-labeled with 0.sub.4 and then fixed with
acid-ethanol, permeabilized and stained with anti-GFAP (IgG). Note
that LNGFR.sup.+ cells (Panel B) are O.sub.4.sup.+ and that most or
all of the O.sub.4.sup.+ cells are also GFAP.sup.+ (Panels E and
F). The quality of the 0.sub.4 staining in (Panel E) appears
different from that in (Panel C) because a redistribution of the
antigen occurs following acid-ethanol fixation. In Panel C, the
flattened O.sub.4.sup.+ cells are more weakly stained for LNGFR
(Panel B). Such flattening is indicative of myelination, and is
consistent with the fact that Schwann cells undergoing myelination
down-regulate LNGFR and up-regulate O.sub.4.
Following 5.times.10 days in SCD medium, most or all of the
non-neuronal cells in the clones expressed glial fibrillary acidic
protein (GFAP), an intermediate filament specific to glial cells,
and sulfatide, a cell-surface glycolipid recognized by the
monoclonal antibody O.sub.4. Triple-labeling of such "mature"
clones with polyclonal anti-peripherin and monoclonal 0.sub.4 and
anti-GFAP antibodies revealed that sulfatide and GFAP were not
expressed by the peripherin-positive neurons and that these two
glial markers were coincident in the non-neuronal cell population
(FIG. 6). FIG. 6 shows a clone from a single founder cell in phase
contrast (Panel A) which expresses LNGFR (Panel B). This clone was
allowed to proliferate and differentiate in complete medium
(containing CEE and lacking serum) and then transferred into SCD
medium (containing serum and forskol in). After approximately 10
days, the culture was fixed and triple-labeled with rabbit
anti-peripherin (Panels C and D, in green/yellow), anti-GFAP (IgG)
(Panel C, in red) and O.sub.4 (IgM) (Panel D, blue). Panels C and D
are two separate fields from the same clone.
Although GFAP is expressed by astrocytes and sulfatide is expressed
by oligodendrocytes in the CNS, the co-expression of these two
markers in the same cell is unique to peripheral glial cells
(Jessen, K. R. et al. (1990) Devel. 109:91-103 and Mirsky, R. et
al. (1990) Devel. 109:105-116).
Therefore, these data indicate that single neural crest cells
expressing nestin and LNGFR are able to give rise to clones of
differentiated cells containing both peripheral neurons and glia.
Differentiation to the neuronal phenotype involves both the loss of
LNGFR and nestin expression, and the gain of neuronal markers such
as neurofilament, high PSA-NCAM and peripherin. On the other hand,
in the glial lineage LNGFR and nestin expression persist, and
additional glial markers (GFAP and O.sub.4) are acquired. All
clones that produced neurons and glia also produced at least one
other cell type that did not express any of the differentiation
markers tested; the identity of these cells is unknown. Taken
together, these data establish the multipotency of the rat neural
crest cell identified and isolated by virtue of co-expression of
LNGFR and nestin.
EXAMPLE 4
Self-renewal of Multipotent Neural Crest Cells in vitro
After 10 days in culture in medium supplemented with 10% CEE and on
a FN/PDL substrate, all of the neural crest cell clones that
contained neurons also contained non-neuronal cells expressing
LNGFR and nestin (as described above). In order to determine
whether these cells were immature glia, or multipotent neural crest
cells that had undergone self-renewal, serial subcloning
experiments were performed. FIG. 7 provides a flow chart
summarizing these serial subcloning experiments. In FIG. 7,
"plating medium" refers to complete medium containing CEE and
lacking FCS and "differentiation medium" refers to SCD medium
containing FCS and forskolin.
For serial sub-cloning experiments, clones were harvested and
replated as follows. The primary clones were examined
microscopically to ensure that there were no impinging colonies and
that the whole clone fits within the inscribed circle. Using
sterile technique throughout the procedure, glass cloning cylinders
(3 mm id.) were coated on one end with silicone grease (Dow
Corning) and placed about the primary clone so that the grease
formed a seal through which medium could not pass. The cells were
removed from the cylinder by first treating them with 100 ml of
0.05% Trypsin solution (Gibco) for 3 minutes at 37.degree. C. in a
humidified 5% CO.sub.2 incubator. At room temperature 70 .mu.l of
the trypsin solution was removed and replaced with 70 .mu.l of
complete medium. The cells were resuspended into the 100 .mu.l
volume by vigorous trituration through a pipet tip and the whole
volume was diluted into 5 ml of complete medium. The 5 ml was then
plated onto 1 or 2 60 mm dishes which were placed in a humidified
5% CO.sub.2 incubator for 2 hours at which time the medium was
exchanged for fresh complete medium. Single founders cells were
then identified and allowed to grow into secondary clones as
described below.
Primary clones founded by LNGFR-positive progenitor cells were
allowed to grow for 6 days (FIG. 8, Panel A) on a PDL/FN substrate.
At this time, clones containing LNGFR-positive cells were
identified by live cell surface labeling, and these clones were
then removed from their original plates by trypsinization, as
described above. The dissociated cells were then replated at clonal
density under the same culture conditions as their founder cells.
Individual secondary founder cells were identified by labeling live
cells with 192-Ig and their positions marked (FIG. 8, Panels B and
B' show two individual secondary founder cells; Panels C and C'
show the clonal progeny of these individual cells at day 17). Both
non-neuronal, neurite bearing cells are visible in the clones (FIG.
8, panels C and C').
A clone derived from secondary founder cells, such as that shown in
FIG. 8, was transferred into SCD medium to allow the expression of
Schwann cell markers. After approximately 10 days, the subclone was
fixed, and double-labeled for NF160 and GFAP (FIG. 9, Panel A shows
the clone in phase contrast; Panel B shows labeling with
anti-NF160; Panel C shows labeling with anti-GFAP). The apparent
labeling of neurons in panel C is an artifact due to bleed-through
into the fluorescein channel of the Texas Red fluorochrome used on
the goat anti-rabbit secondary antibody in panel B.
Additionally, following 10 days of secondary culture, living
subclones were scored visually for the presence of neurons and glia
by double labeling with 192-Ig (anti-LNGFR) and 5A5, a monoclonal
antibody to high PSANCAM.
Single neural crest cells isolated from primary clones were able to
proliferate and generate clones containing both neurons and
non-neuronal cells, probably glia. Quantitative analysis of clones
derived from 16 different primary and 151 secondary founders after
ten days in plating medium indicated that over 30% of the total
secondary founder cells gave rise to clones containing neurons (N),
glia (G) and other (O) cells (Table I, N+G+O). Of the remaining 70%
of the founder cells, however, almost 50% failed to form clones and
died; thus of the clonogenic (i.e., surviving) founders, 54% were
of the N+G+O type (Table I). To confirm that these mixed clones
indeed contained glia or glial progenitors, they were transferred
to SCD medium and allowed to develop for an additional 7 days, then
fixed and double-stained for neurofilament and GFAP expression. As
was the case for the primary clones, this treatment caused
expression of GFAP in a high proportion of non-neuronal cells in
the clones (FIG. 9), confirming the presence of glia. These data
indicate that primary neural crest cells are able to give rise at
high frequency to progeny cells retaining the multipotency of their
progenitors, indicative of self renewal. However, in several cases
secondary clones containing only neurons were found (Table I, N
only), and many of the secondary clones contained glia and other
cells but not neurons (Table I, G+O). This observation suggests
that in addition to self-renewal, proliferating neural crest cells
may undergo lineage restriction in vitro as well to give rise to
glial or neuronal progenitor cells which are characterized by the
capacity to divide and self-renew but are restricted to either the
neuronal or glial lineage.
TABLE I
__________________________________________________________________________
Sub-Clone Phenotype total#(%) Primary # of 2.degree. No clone Clone
ID Founders N only N + G + O G + O O found
__________________________________________________________________________
1.1 21 0 15 (71) 0 0 6 (29) 1.18 6 0 1 (17) 1 (17) 2 (33) 2 (33)
1.24 5 1 (20) 0 1 (20) 2 (40) 1 (20) 2.6 7 0 0 1 (14) 1 (14) 5 (72)
2.18 7 0 0 1 (14) 0 6 (86) 3.14 20 0 2 (10) 4 (20) 0 14 (70) 3.18 4
0 1 (25) 0 0 3 (75) 4.5 1 0 1 (100) 0 0 0 4.8 9 0 0 1 (11) 2 (22) 6
(67) 4.14 10 0 2 (20) 3 (30) 1 (10) 4 (40) 5.2 15 1 (7) 8 (53) 0 0
6 (40) 6.1 13 0 2 (15) 2 (15) 0 9 (70) 6.2 17 1 (6) 2 (12) 4 (24) 0
10 (58) 6.17 2 0 1 (50) 0 0 1 (50) 8.2 5 0 4 (80) 0 0 1 (20) 8.5 9
0 4 (44) 0 0 5 (56) Mean .+-. s.e.m. % total 2.1 .+-. 1.3 31 .+-.
7.9 10 .+-. 2.6 7.4 .+-. 3.3 49 .+-. 6 founders % clono- 3.1 .+-.
1.8 54 .+-. 11 29 .+-. 8 15 .+-. 6 genic founders
__________________________________________________________________________
EXAMPLE 5
Substrate Composition Influences the Developmental Fate of
Multipotent Neural Crest Cells
The foregoing experiments indicate that neural crest cells grown on
a PDL/FN substrate generate clones containing both peripheral
neurons and glia. When the same cell population is grown at clonal
density on a substrate containing FN only, the resulting clones
contain glia and "other" cells but never neurons (FIGS. 10 and 11,
Panels D,E,F). FIG. 10 provides a flow chart summarizing the
following experiments which demonstrate the substrate effect on the
fate of mammalian neural crest cells. FIG. 11 shows the
immunoreactivity of cells stained for various markers.
On FN alone, G+O clones are obtained containing non-neuronal cells
expressing high levels of LNGFR immunoreactivity, but neither
NCAM.sup.+ nor neurite-bearing cells (FIG. 11, panels E,F). By
contrast on PDL/FN, the clones contain both LNGFR.sup.+, NCAM
non-neuronal crest cells and LNGFR, NCAM.sup.+ neurons (FIG. 11,
panels B,C). Quantification indicated that on FN alone, 70-80% of
the clones are of the G+O phenotype and none of the N+G+O phenotype
(FIG. 12, panel A), whereas on PDL/FN 60% of the clones are of the
N+C+O and only 20% are of the GAO phenotype (FIG. 12, panel B).
These data indicate that the composition of the substrate affects
the phenotype of neural crest cells that develop in culture.
To rule out the possibility that the foregoing results could be
explained simply by the failure of neurogenic crest cells to adhere
and survive on a FN substrate, a different experiment was performed
in which all the crest cells were initially cloned on a FN
substrate.
FIG. 13 provides a flow chart summarizing these experiments. These
experiments were performed to demonstrate that differences in
attachment and/or survival do not account for differences in
eventual clone composition. Subsequently, one group of cells was
exposed to PDL as an overlay in liquid media (0.05 mg/ml) after 48
hrs, while a sister culture was retained on FN alone as a control
(FIG. 13). Clones expressing LNGFR were identified by live cell
surface labeling at the time of the PDL overlay and the development
of only LNGFR.sup.+ clones was further monitored. After two weeks,
the cultures were transferred to SCD medium for an additional 10
days of culture, and their phenotypes then scored as previously
described.
By contrast to clones maintained on FN, where no neurons developed,
many of the clones exposed to a PDL overlay contained neurons at
the end of the culture period (FIG. 14, panel A). Moreover,
virtually none of the clones were of the G+O phenotype after the
PDL overlay. These data indicate that an overlay of PDL is able to
alter the differentiation of neural crest cells even if they are
initially plated on an FN substrate. Moreover, they suggest that at
least some of the N+G+O clones derived by conversion of founder
cells that would have produced G+O clones on FN. However, because
of the increased cytotoxicity obtained from the PDL overlay, it was
not possible to rule out the possibility that many of the cells
that would have produced G+O clones simply died. To address this
issue, the PDL overlay was performed on a parallel set of cultures
at day 5 rather than at 48 hrs. Under these conditions, virtually
all of the LNGFR.sup.+ clones survived and differentiated. 60% of
these clones contained neurons, whereas 35% contained GAO (FIG. 14,
panel B). By contrast, greater than 90% of the clones maintained on
FN developed to a G+O phenotype. Since little or no clone death was
obtained under these conditions, and since a majority of the clones
contained neurons following the PDL overlay at day 5, these data
suggest that PDL converts presumptive G+O clones into N+G+O clones.
However the fact that 35% of the clones became G+O following PDL
overlay at days, whereas virtually none did so when the overlay was
performed at 48 hrs (FIG. 14, compare G+O, hatched bars, in panels
A and B), suggests that some clones might become resistant to the
effect of PDL between 48 hrs and days.
EXAMPLE 6
Substrate Influences Latent Developmental Potential of Neural Crest
Cells
To demonstrate more directly that the substrate can alter the
developmental fate of neural crest cells, a serial subcloning
experiment was performed. Clones were established on FN, and after
5 days the progeny of each clone were subdivided and cloned onto
both FN and PDL/FN substrates. Following 10 days of culture in
standard medium, the clones were shifted to SCD medium for an
additional week to ten days and then fixed, stained and scored for
the presence of neurons and Schwann cells. Five of seven primary
clones founded on FN gave rise to secondary clones containing
neurons when replated onto a PDL/FN substrate at days (Table II).
On average, 57.+-.17% of the secondary clones contained neurons. By
contrast, none of the sister secondary clones replated onto FN
contained neurons (Table II). These data confirm that the PDL/FN
substrate is able to alter the fate of neural crest cell clones
initially grown on FN. They also reveal that the "neurogenic
potential" of neural crest cells is retained, at least for a period
of time, on FN even though overt neuronal differentiation is not
observed. This suggested that FN is non-permissive for overt
neuronal differentiation under these culture conditions. In support
of this idea, when primary clones established on PDL/FN were
replated onto FN, none of the secondary clones contained neurons,
whereas 100% (5/5) of the primary clones gave rise to
neuron-containing secondary clones when replated onto PDL/FN (Table
II). Moreover, on average 93.+-.7% of the secondary clones derived
from each primary clone contained neurons on PDL/FN, indicating
that most or all of the clonogenic secondary crest cells retained
neurogenic potential under these conditions.
While this experiment indicated that at least some neural crest
clones retain neurogenic potential on FM, not all clones exhibited
this capacity. This could indicate a heterogeneity in the
clonogenic founder cells that grow on FN, or it could indicate a
progressive loss of neurogenic potential with time in culture on
FM. To address this issue, a second experiment was performed in
which primary clones were replated at day 8 rather than at day 5.
In this case, a more dramatic difference was observed between
primary clones established on FM versus on PDL/FN. Only 1/6 primary
FM clones replated at day 8 gave rise to any secondary clones
containing neurons on PDL/FN, and in this one case only 17% of the
secondary clones contained neurons (Table II). By contrast, 6/6
primary PDL/FN clones gave rise to neuron-containing secondary
clones when replated on PDL/FN at day 8, and 52+7% of these
secondary clones contained neurons (Table II). These data suggest
that neurogenic potential is gradually lost by neural crest cells
cultured on FM, but retained to a much greater extent by the same
cells grown on PDL/FN. Thus the composition of the substrate
influences not only the overt differentiation of the neural crest
cells, but also their ability to maintain a latent developmental
potential over multiple cell generations.
TABLE II ______________________________________ % % Neuronal
1.degree. Substrate FN pDL/FN 2.degree. Substrate FN pDL/FN FN
pDL/FN Neuronal ______________________________________ Day 5 0/7
5/7 57 .+-. 17 0/5 5/5 93 .+-. 7 Replating Day 8 0/6 1/6 17 0/6 6/6
52 .+-. 7 Replating ______________________________________
EXAMPLE 7
Identification of Neural Crest Stem Cells by Transplantation
Neural crest stem cells are identified by two general criteria: by
their antigenic phenotype, and by their functional properties.
These functional properties may be assessed in culture (in vitro),
as described above, or they may be assessed in an animal (in vivo).
The above examples described how the self-renewal and
differentiation of neural crest stem cells can be assayed in vitro,
using clonal cell cultures. However, these properties may also be
determined by transplanting neural crest cells into a suitable
animal host. Such an assay requires a means of delivering the cells
and of identifying the transplanted cells and their progeny so as
to distinguish them from cells of the host animal. Using standard
techniques, it is possible to deliver neural crest cells to a
developing mammalian or avian embryo or to any tissue or
compartment of the adult animal (e.g., brain, peritoneal cavity,
etc.).
For example, neural crest cell cultures are prepared as described
earlier. After a suitable period in primary or secondary culture,
neural crest cells are identified by live cell-labeling with
antibodies to LNGFR, and removed from the plate using trypsin and a
cloning cylinder, as described in previous examples. The cells are
diluted into serum-containing medium to inhibit the trypsin,
centrifuged and resuspended to a concentration of 10.sup.6
-10.sup.7 cells per milliliter. The cells are maintained in a
viable state prior to injection by applying them in small drops
(ca. 10 .mu.l each) to a 35 mm petri dish, and evaporation is
prevented by overlaying the droplets with light mineral oil. The
cells are kept cold by keeping the petri dishes on ice. For
injections into mouse embryos, pregnant mothers at embryonic day
8.5-9.0 are anaesthetized and their uterus exposed by an incision
into the abdomen. Neural crest cells are drawn into a sharpened
glass micropipette (with a sealed tip and hole in the side to
prevent clogging during penetration of tissues) by gentle suction.
The pipette is inserted into the lower third of the deciduum and a
volume of approximately 0.5 .mu.l is expelled containing
approximately 1000 cells. The micropipette is withdrawn and the
incision is sutured shut. After an additional 3-4 days, the mother
is sacrificed, and individual embryos are removed, fixed and
analyzed for the presence and phenotype of cells derived from the
injected neural crest cells.
To identify the progeny of the injected cells, it is necessary to
have a means of distinguishing them from surrounding cells of the
host embryo. This may be done as follows: rat neural crest cells
are injected into a mouse embryo (following suitable
immunosuppression of the mother or using a genetically
immunodeficient strain such as the SCID strain of mice), the
injected cells are identified by endogenous markers such as Thyl or
major histocompatibility complex (MHC) antigens using monoclonal
antibodies specific for the rat Thyl or MHC antigens.
Alternatively, an exogenous genetic marker is introduced into the
cells prior to their transplantation as a means of providing a
marker on or in the injected cells. This is as follows: neural
crest cells in culture are incubated with a suspension of
replication-defective, helper-free retrovirus particles harboring
the lacZ gene, at a titer of 10.sup.5 -10.sup.6 pfu/ml in the
presence of 8 .mu.l/ml polybrene for four hours. The cells are then
washed several times with fresh medium and prepared for injection
as described above. The harvested embryos are then assayed for
expression of .beta.-galactosidase by whole mount staining
according to standard procedures. The blue cells (indicating
expression of the lacZ gene) will correspond to the progeny of the
injected neural crest cells. This procedure can be applied to any
tissue or any stage of development in any animal suitable for
transplantation studies. Following whole-mount staining, embryos
bearing positive cells are embedded in freezing medium and
sectioned at 10-20 .mu.m on a cryostat. Sections containing blue
cells are selected, and then counterstained for markers of mature
neurons and glia using specific antibodies, according to standard
techniques, and immunoperoxidase or alkalinephosphatase
histochemistry. The identification of lacZ+ (blue) cells expressing
neuronal or glial markers indicates that the progeny of the
injected neural crest cells have differentiated appropriately.
Thus, this technique provides a means of identifying mammalian
neural crest stem cells through transplantation studies to reveal
the function of said stem cells.
EXAMPLE 8
Genetic-Engineering of Neural Crest Stem Cells (NCSCs)
A. Retroviral infection of NCSCs
In this method, NCSCs are infected with a replication-incompetent,
recombinant retrovirus harboring the foreign gene of interest. This
foreign gene is under the control of the long terminal repeats
(LTRs) of the retrovirus, in this case a Moloney Murine Leukemia
Virus (MoMuLv) (Cepko et al. (1984) Cell 37:1053-1062).
Alternatively, the foreign gene is under the control of a distinct
promoter-enhancer contained within the recombinant portion of the
virus (i.e., CMV or RSV LTR). In this particular example, the E.
coli .beta.-galactosidase gene was used, because it provides a blue
histochemical reaction product that can easily be used to identify
the genetically-engineered cells, and thereby determine the
transformation efficiency.
Rat NCSC cultures were established as described above. Twenty-four
hours after replating, the cells were exposed to a suspension of
.beta.-galactosidase-containing retrovirus (Turner et al. (1987)
Nature 328:131-136) with a titer of approximately 10.sup.5
-10.sup.6 pfu/ml in the presence of 8 .mu.g/ml polybrene. Following
a 3 hr exposure to the viral suspension, the cultures were rinsed
and transferred into standard medium. After three days of growth in
this medium, the transformed cells were visualized using the X-gal
histochemical reaction (Sanes et al. (1986) EMBO J. 5:3133-3142)
FIG. 15, Panel A shows the NCSC culture three days after infection
with the lacZ containing retrovirus, after fixation and staining
using the X-gal reaction. .beta.-galactosidase-expressing cells are
indicated by the solid arrows. Non-expressing cells in the same
microscopic field are visualized by phase contrast microscopy (B),
and are indicated by open arrows. The blue,
.beta.-galactosidase.sup.+ cells represented approximately 5-10% of
the total cells in the culture as visualized by phase-contrast
microscopy (FIG. 15, Panel B).
B. Calcium-Phosphate-Mediated Transfection of NCSCs
In this method, NCSCs are transfected with an expression plasmid
using the calcium phosphate method (Wigler et al. (1979) Proc.
Natl. Acad. Sci. USA 76:1373-1376). As in the previous example, the
.beta.-galactosidase gene was used to facilitate visualization of
the transfected cells.
In this case, the vector pRSVlacZ was used, in which the
.beta.-galactosidase gene (lacZ) is under the control of the Rous
Sarcoma Virus (RSV) LTR, and the SV40 intron and poly A-addition
site are provided at the 3' end of the gene (Johnson et al. (1992)
Proc. Natl. Acad. Sci. USA 89:3596-3600).
NCSCs were established in 35 mm tissue culture dishes. 24 hr after
plating, a calcium phosphate precipitate containing approximately
20 .mu.g/ml of pRSVlacZ was prepared. 123 .mu.l of this precipitate
was added to each dish, and incubated at room temperature for 20
minutes. Two ml of standard medium containing 30 .mu.M chloroquine
was then added to each dish and incubation was continued overnight
at 37.degree. C. The next day, the medium was replaced and
incubation continued for a further two days. The cultures were then
fixed and assayed for .beta.-galactosidase expression by the
standard X-gal reaction. Approximately 10% of the NCSCs expressed
the lacZ reaction product.
C. Immortalization of NCSCs
NCSC cultures are established as described above. The cultures are
exposed, in the presence of 8 .mu.g/ml polybrene, to a suspension
of retrovirus harboring an oncogene preferably selected from the
immortalizing oncogenes identified herein. These retroviruses
contain, in addition to the oncogene sequences, a gene encoding a
selectable marker, such as hisD, driven by the SV40 early
promoter-enhancer (Stockschlaeder, M. A. R. et al. (1991) Human
Gene Therapy 2:33). Cells which have taken up the hisD gene are
selected for by growth in the presence of L-histidinol at a
concentration of 4 mM. Alternatively, selection can be based upon
growth in the presence of neomycin (500 .mu.g/ml). NCSCs are
infected with the above retroviruses which are concentrated to a
titer of greater than 10.sup.6 pfu/ml by centrifugation. The virus
is applied to the cells in two sequential incubations of 4-8 hours
each in the presence of 8 .mu.g/ml polybrene.
Following infection, the cells are grown in the presence of 4 mM
L-histinol or 500 .mu.g/ml neomycin (G418) for 5-10 days. Cells
which survive the selection process are screened for expression of
LNGFR by live-cell labeling using the monoclonal antibody 192 Ig as
described above. Colonies containing a homogeneous population of
LNGFR+ cells are cloned using a cloning cylinder and mild
trypsinization, and transferred into duplicate FN/pDL-coated
96-well plates. After a short period of growth, one of the plates
is directly frozen (Ramirez-Solis, R. et al. (1992) Meth. Enzymol.,
in press). The cells in the other plate are replated onto several
replicate 96-well plates, one of which is maintained for carrying
the lines. The cells on the other plates are fixed and analyzed for
the expression of antigenic markers. Successful immortalization is
indicated by (1) the cells homogeneously maintain an antigenic
phenotype characterized by LNGFR+, nestin+, lin- (where "lin"
refers to lineage markers characteristic of differentiated neuronal
or glial crest derivatives, including neurofilament, peripherin, hi
PSA-NCAM, GFAP, O4 and P.sub.o); and (2) the cell population is
phenotypically stable over several weeks of passage (as defined by
lack of differentiation to morphologically- and
antigenically-recognizable neurons and/or glia). The ability of the
lines to differentiate is tested by transferring them to conditions
that promote differentiation (omission of CEE in the case of
neurons and addition of serum and 5 .mu.M forskolin for Schwann
cells). Maintenance of the ability to differentiate is a desirable,
although not necessary, property of the constitutively-immortalized
cells.
EXAMPLE 9
Generation of Monoclonal Antibody to Mouse LNGFR
Mouse monoclonal antibodies specific to LNGFR from primates (Loy et
al. (1990), J. Neruosci. Res. 27:657-664) and rat (Chandler et al.
(1984) J. Biol. Chem. 259:6882-6889) have been produced. No
monoclonal antibodies to mouse LNGFR have been described. We have
produced rat monoclonal antibodies to mouse LNGFR. These antibodies
recognize epitopes present on the surface of living cells such as
Schwann cells, making them suitable for use in immunologic
isolation of multipotent neural stem cells (such as neural crest
stem cells) and their differentiated derivatives (as well as neural
progenitor cells from the CNS) from murine species. The isolation
of such cells from mice is particularly desirable, as that species
is the experimental organism of choice for genetic and
immunological studies or human disease.
To generate monoclonal antibodies to mouse LNGFR, a genomic DNA
fragment encoding the extracellular domain (ligand binding domain)
of that protein was expressed in E. coli, as a fusion protein with
glutathione-S-transferase (Lassar et al. (1989) Cell 58:823-831).
Briefly, a probe for the extracellular domain based on either of
the known DNA sequences for rat and human LNGFR is used to screen a
mouse genomic library. A cloned insert from a positively
hybridizing clone is excised and recombined with DNA encoding
glutathione with appropriate expression regulation sequences and
transfected into E. coli. The fusion protein was affinity-purified
on a glutathione-Sepharose column, and injected into rats. Sera
obtained from tail bleeds of the rats were screened by
surface-labeling of live Schwann cells isolated from mouse sciatic
nerve by standard procedures (Brockea et al. (1979) In Vitro
15:773-778. Surface labeling was with labelled goat anti-rat
antibody Following a boost, fusions were carried out between the
rat spleen cells and mouse myeloma cells. Supernatants from the
resulting hybridoma cultures were screened using the live Schwann
cell assay. Positive clones were re-tested on NIH 3T3 fibroblasts,
a mouse cell line that does not express LNGFR, and were found to be
negative. The use of this live cell assay ensures that all
antibodies selected are able to recognize LNGFR on the surface of
living cells. Moreover the assay is rapid, simple and more
efficient than other assays such as ELISA, which require large
quantities of purified antigen.
Approximately 17 independent positive hybridoma lines were
identified and subcloned. An example of the results obtained with
the supernatant from one such line 19 shown in FIG. 16. A culture
of mouse sciatic nerve Schwann cells was labeled with one of the
rat anti-mouse LNGFR monoclonal antibodies and counterstained with
DAPI to reveal the nuclei of 611 cells. The left panel (A) shows
that most of the cells are labeled on their surface with the
anti-LNGFR antibody (red staining; solid arrows), the right panel
(B) reveals all the cell nuclei on the plate, and shows a few cells
not labeled by the anti-LNGFR antibody (blue staining; open arrows;
compare to left panel). These unlabeled cells most likely represent
contaminating fibroblasts which are known not to express LNGFR.
These cells provide an internal control which demonstrates the
specificity of the labeling obtained with the anti-LNGFR
antibody.
EXAMPLE 10
O Cells are Smooth Muscle Cells
To determine whether O cells could be smooth muscle cells, cultures
of neural crest cells containing these cells were stained with a
monoclonal antibody to smooth muscle actin (SMA), a marker of
smooth muscle cells (Skalli et al (1966) J. Cell Biol.
103:2787-2796). The cultures were counter-stained with anti-p75to
identify the neural crest stem cells. The anti-SMA antibody labeled
a significant number of cells (FIG. 17B, open arrows), and these
cells did not express p75on their surface and were clearly distinct
from the p75expressing neural crest stem cells (FIG. 17B, closed
arrow). However, clonal analysis indicated that both p75.sup.+,
SMA.sup.- cells and p75.sup.-, SMA.sup.+ cells derived from a
p75.sup.+ neural crest stem cell progenitors (see below).
To establish that individual neural crest stem cells could generate
neurons, glia and smooth muscle cells, a clonal analysis was
performed. Individual p75+ neural crest stem cells were identified
and allowed to develop for two weeks in culture. The resultant
clones were then fixed and triply-labeled with antibody to
peripherin (to detect neurons), GFAP (to detect glia) and SMA (to
detect smooth muscle cells). As shown in FIG. 18, within the same
clone it was possible to identify neurons (FIGS. 18A, 18B,
arrowhead), glia (FIGS. 18C, open arrows) and smooth muscle cells
(FIG. 18C, closed arrow), confirming that the neural crest stem
cell is able to generate all three lineages in our culture
system.
The foregoing experiments were carried out in standard medium (SM)
lacking fetal bovine serum. Previously, we observed that the
addition of fetal bovine serum to this medium at early times of
culture resulted in the extinction of LNGFR expression. Taken
together with the foregoing observation that SMA.sup.+ cells are
LNGFR, we asked whether cells grown in SM+fetal bovine serum
expressed smooth muscle markers. The results indicate that
virtually all cells obtained in SM+fetal bovine serum express high
levels of SMA (FIGS. 20A, 20B). To further establish their identity
as smooth muscle cells, these cells were also stained with two
other markers of smooth muscle: desmin (Lazarides, et al (1978)
Cell 14:429-438) and calponin (Gimona et al (1990) FEBS Lett.
274:159-162). The SMA+ cells were also labeled by anti-desmin
antibody (FIG. 3C) and by anti-calponin (FIGS. 3A, B). These data
confirm that the O cells are indeed smooth muscle cells, and also
show that fetal bovine serum contains one or more substances able
to drive virtually all neural crest stem cells into the smooth
muscle lineage.
Differentiated smooth muscle cells have been isolated and cultured
from the vasculature, for example, Chamley-Campbell et al (1990)
Phys. Rev. 59:1-61, but previously it has not been possible to
obtain the de novo differentiation of such cells from an
undifferentiated progenitor. The data presented above identify
neural crest stem cells as progenitors of smooth muscle, as well as
of neurons and glia, and indicate that they can be induced to
differentiate to smooth muscle in culture using fetal bovine serum.
Such differentiation occurs at the expense of neuronal and glial
differentiation, which does not occur in the present of fetal
bovine serum (Stemple et al (1992), Id.). Thus, neural crest stem
cells should be useful for identifying smooth muscle
differentiation factors present in fetal bovine serum, as well as
for identifying other growth, survival or differentiation factors
for smooth muscle present in other sources.
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